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Y. 10460 OFFICERS Laurance S. Rockefeller Robert G. Goelet Howard Phipps, Jr. Chairman, Board of Trustees President Chairman, Executive Committee Henry Clay Frick, II George F. Baker, Jr. John Pierrepont Augustus G. Paine, 11 Vice-President Vice-President Treasurer Secretary Edward R. Ricciuti Joan Van Haasteren Editor & Curator, Associate Editor Publications & Public Relations William G. Conway Donald R, Griffin Hi (,H B. House EDITORIAL COMMITTEE Robert G. Goelet Chairman F. Wayne King James A. Oliver Peter R. Marler Edward R. Ricciuti Ross F. Nigrelli George D. Ruggieri, S.,1. William G. Conway General Director ZOOLOGICAL PARK William G. Conway . . . Director ct Curator, Ornithology Hugh B. House .... Ctirator, Mammalogy James G. Doherty .... Assistant Curator, Mammalogy Joseph Bell . . Associate Curator, Ornithology Donald F. Bi nning . Assistant Curator, Ornithology F. Wayne King .... Curator, Herpetology John L. Behler, Jr Herpetologist Walter Auffenberg . Robert A. Brown . . Joseph A. Davis . . Emil P. Dolensek John M. Budinger . Ben Sheffy .... William Bridges . Cnn . Research Associate in Herpetology Assistant Ctirator, Animal Departments . Scientific Assistant to the Director Veterinarian . Constiltant, Pathology , Constiltant, Nutrition '• of Publications Emeritus AQUARIUM James A. Oliver Director John Clark Curator Christopher W. Coates . . . Director Emeritus Louis Mowbray . Research Associate iiiField Biology lay Hyman Constiltant Veterinarian OSBORN LABORATORIES OF MARINE SCIENCES Ross F. Nigrelli . . . Director and Pathologist George D. Ruggieri, S.J. . . Assistant Director & Experimental Embryologist Martin F. Stempien, Jr. . . . Assistant to the Director & Bio-Organic Chemist Eli D. Goldsmith .... Scientific Consultant William Antopol . . . Research Associate in Comparative Pathology C. M. Breder. Jr. . . . Research Associate in Ichthyology Harry A. Charipper . . 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Research Zoologist Alan Fill Research Associate O. Marcus Buchanan . . . Resident Director, William Beebe Tropical Research Station Contents Issue 1. May 1, 1970 PAGE 1. Sex Determination and the Restriction of Sex-Linked Pigment Patterns to the X and Y Chromosomes in Populations of a Poeciliid Fish, Xiphophonis maculatus, from the Belize and Sibun Rivers of British Honduras. By Klaus D. Kallman. Plates I-II; Text-figure 1 1 Issue 2. October 23, 1970 2. Metabolism, Energetics, and Thermoregulation During Brooding of Snakes of the Genus Python (Reptilia, Boidae). By Allen Vinegar, Victor H. Hutchison, and Herndon G. Dowling. Plates I-II; Text-figures 1-24.... 19 Issue 3. December 15, 1970 3. A Preliminary Study on the Immobilization of the Asiatic Elephant {Elephas maximus) Utilizing Etorphine (M-99). By C. W. Gray and A. P. W. Nettashinghe. Plates I-II 51 4. Epizootics in Yellowtail Flounder, Limanda ferruginea Storer, in the Western North Atlantic Caused by Ichthyophonus, an Ubiquitous Parasitic Fungus. By George D. Ruggieri, S.J., Ross F. Nigrelli, P. M. Powles, AND D. G. Garnett. Plates I-X; Text-figure 1 57 Issue 4. January 19, 1971 5. Gonadotrophin in the Urine of a Pregnant Indian Elephant — A Case Report. By Eho Fujimoto, Natsuki Koto, Tatsuo Imori, and Sanenori Nakama. Plates I-II 73 Index to Volume 55 80 ZOOLOGICA SCIENTIFIC CONTRIBUTIONS OF THE NEW YORK ZOOLOGICAL SOCIETY VOLUME 55 • ISSUE 1 • SPRING, 1970 PUBLISHED BY THE SOCIETY The ZOOLOGICAL PARK, New York Contents PAGE 1. Sex Determination and the Restriction of Sex-linked Pigment Patterns to the X and Y Chromosomes in Populations of a PoecUiid Fish, Xipho- phorus maculatus from the Belize and Sibun Rivers of British Honduras. By Klaus D. Kallman. Plates I-II; Text-figure 1 1 Manuscripts must conform with Style Manual for Biological Journals (American Institute of Biological Sciences). All material must be typewritten, double-spaced. Erasable bond paper or mimeograph bond paper should not be used. Please submit an original and one copy of the manuscript. ZooLOGtCA is published quarterly by the New York Zoological Society at the New York Zoological Park. Bronx Park. Bronx, N. Y. 10460. and manuscripts, subscriptions, orders for back issues and changes of address should be sent to that address. Subscription rates: $6.00 per year; single numbers, $1.50, unless otherwise stated in the Society's catalog of publications. Second-class postage paid at Bronx, N. Y. Published May 1, 1970 © 1970 New York Zoological Society. All rights reserved. 1 Sex Determination and the Restriction of Sex-linked Pigment Patterns to the X and Y Chromosomes in Populations of a Poeciliid Fish, Xiphophoriis maculatus, from the Belize and Sibun Rivers of British Honduras' Klaus D. Kallman= { Plates I-II; Text-figure 1 ) X. maculatus is polymorphic for sex chromosomes and sex-linked pigment patterns. Females of natural populations may be of the genotype WY, WX, or XX and males XY or YY. Fish from the two rivers were tested for their sex-genotypes, because earlier but limited data had indicated that the X is absent from rivers in British Honduras. Of 8 males and 30 females tested, X chromosomes were only found in one male (XY) and female (XX). One WY female was fertilized by an XY male, before she was col- lected. Of the 29 females with a W, all but two exhibited one or more sex-linked pigment pattern controlled by at least three loci. The 27 females possessed a total of 41 factors. None was W-linked. In preserved collections from these rivers, the frequency of males with macromelanophore patterns was two to three times that of females. This difference is in good agreement with the hypothesis that in natural populations the W chromosome does not carry pigment factors. This is not true for the X chromosome. Since crossing over beween the W and Y has been observed in the laboratory, it must also occur in natural populations. In the absence of selection, crossing over should tend to equalize the frequency of marked W and Y chromosomes. A selective advantage is postulated for high coloration in males and a disadvantage in females. The significance of the W as a vehicle for strict maternal transmission of characters is discussed. Introduction The southern platyfish, Xiphophoriis maculatus, has been the subject of many investigations, because of its unusual pig- mentary and sex chromosome polymorphism. In X. maculatus, which ranges from southern Mexico near Veracruz to British Honduras, females may be of the sex genotypes XX, WX, or WY and males may be XY or YY. The sex chromosomes have not been identified cyto- logically, but an abundance of data concerned with sex ratios and the inheritance of sex-linked pigment patterns attest to the reality of the W, 'This investigation was supported in part by a grant, CA 06665, from the National Cancer Institute, U. S. Public Health Service. “Genetics Laboratory, Osborn Laboratories of Marine Sciences, New York Zoological Society, Brooklyn, N.Y. 11224. X, and y chromosomes (Bellamy 1922, 1924, 1928; Bellamy and Queal 1951; Fraser and Gordon 1929; Gordon 1927, 1937, 1946, 1947, 1952; Kallman 1965). The geographical distribution of the W and X chromosome has been the subject of some con- troversy and misunderstanding. Based upon experiments with platyfish obtained through commercial sources it was thought that sex de- termination in this species was of the WY-YY type (Bellamy 1922, 1924, 1928; Breider 1942; Gordon 1927, 1937; Kosswig 1938). In 1947 Gordon discovered the X chromosome in Mexi- can populations of platyfish and later stated that X. maculatus with an XX-XY mechanism in- habited rivers in Mexico and were geographically isolated from populations with a WY-YY system in British Honduras (Gordon 1954). This theory has been widely accepted in many review papers and monographs on sex deter- 1 2 Zoologica: New York Zoological Society [55; 1 mination, but it is now outdated. More recent experiments with fish collected in several major drainages have shown that W and X chromo- somes occur together in roughly 70 percent of the total known range of this species (Kallman 1965). The two female determining chromo- somes, W and X, have been found in the Rio Grijalva, Rio de la Pasion (Rio Usumacinta system), Lake Peten, and Rio Hondo. Only in populations of the Rio Jamapa, Papaloapan, and Coatzacoalcos in the western part of the species range, has the W not been demonstrated; the X is not known from the New River and Belize River in British Honduras. Because rela- tively few fish were tested, the failure to demon- strate the IV OT X chromosomes from these rivers may be due to a sampling error. Kallman (1965) and Kallman and Atz (1967) have sug- gested that the sex chromosome mechanism of X. maciilatiis probably has arisen from a XX- XY type as is present today in X. v. variants, X. xiphidiiim, X. milleri, and X. p. nigrensis. The sex chromosomes of the four species are homologous (Kallman and Atz 1967; Zander 1968). Some sort of selective advantage must have been and perhaps still is associated with the W chromosome, since it is widespread today. To understand the evolution of the sex de- termining system of X. maciilatiis, it is important to establish whether the X is absent from any major river system. Such populations could have arisen as the result of the replacement of the X by the W; or they could have been founded by fish already possessing the WY-YY mecha- nism. This paper is concerned with sex deter- mination of the platyfish populations inhabiting the Belize River and Sibun River of British Honduras. Material and Methods The three collecting stations can be found on Text-fig. 1. Fish from the Sibun River were collected at a locality called Freetown, in a small weedy pond on the left side of the dirt track that branches off the Western Highway about 2 km beyond Hattieville. Fish were caught by repeated sweeps with a 10 feet long, 4 feet wide (one quarter inch mesh) nylon seine along 20 meters of shore line. Because of dense shrubbery other parts of the bank were not accessible. The location, hereafter designated Freetown, was visited on Jan. 20 and 23, 1966. The data pre- sented in Table 7 represent the combined count of the two collections. The ten females and two males which were brought alive to the Genetics Laboratory were given pedigree number 1899 (Table 1 and Table 2) and with the exception of male 1899-12 (Table 1) were collected dur- ing the second visit. Text-fig. 1. Map of British Honduras showing the environs of Belize City. Collecting stations in the Belize River drainage are on the north side of the road leading to Bermudian Landing (above the “u” of Bermudian) and Gabourel Creek near Stanley airport. Collecting station for the Sibun River is at Freetown. Fish from the Belize River were caught in a shallow, broad lagoon, located in a cow pasture on the right side of the dirt road running from the town of Boom towards the ferry crossing at Bermudian Landing. The exact location is “Mamre Farm,” approximately 2 km east of the ferry. The data in Table 7 represent the com- bined count of two collections made on Jan. 21 and 24. The fish used for breeding purposes were collected on Jan. 24 and were assigned pedigree number 1900 (Table 1 and Table 3). This col- lecting station will be referred to as “Bermudian Landing.” The second collecting spot along the Belize River was Gabourel Creek, a ditch one to two meters deep that extends from the eastern limit of the main runway of the Belize airport (Stanley Field) towards the Belize River 1 km away. All fish were caught on Jan. 22, 1966, in close proximity to where the creek runs below the access road to Stanley Field; those kept alive for genetic experiments were given pedigree number 1901 (Table 1 and Table 4). 1970] Kallman: Sex Determination and the Restriction of Sex-linked Pigment Patterns 3 The sex genotypes of the males collected in the Sibun River and Belize River were identified by mating them to XX females of the Genetics Laboratory reference stocks (Kallman 1965). Males of the genotype YY sire all male broods. Males with the XY constitution give rise to off- spring of both sexes in equal frequency; the paternal sex-linked pigment pattern, if present, is inherited by one sex only. The sex chromosomes of the females from the two rivers were identified by either one of two methods. Some fish were mated to XY males of the reference stocks in which the X and Y chromosomes were differently marked. Females with the WY genotype give rise to a 1 ; 1 sex ratio with both the X- and T-linked paternal pigment patterns exhibited by one half of the offspring of both sexes. A 1 : 1 sex ratio is also obtained with XX females, but the X- linked pattern of the male parent is inherited by females and the T-linked pattern by males only. WX females produce broods with a ratio of 3 $9: 1 $$: the A-linked paternal pattern is in- herited by two-thirds of the female but none of the male offspring; the T-linked pattern of the male parent is inherited by every male offspring but by only one third of the females. Other females of the Sibun River and Belize River were mated to males known to be YY. Females with the genotype XX give rise to all male broods. Both WY and WX females give rise to a 1:1 sex ratio, but can be told apart by mating a male offspring of the Fj generation to a XX female of the reference stocks. The male offspring of WX females are XY and sire broods that consist of both sexes; those of WY females are YY and give rise to all male progeny. The identification of the sex genotypes of newly-collected females is greatly facilitated by the presence of sex-linked pigment patterns. Therefore most fish shipped to the Genetics Laboratory were marked. In this respect the breeding data reported in this paper represent a biased sample. In the field the fish of each seine haul were immediately examined by the author for pigment patterns. Fish were kept alive if they exhibited a pattern or combination of patterns net yet present in the collection. Usually not more than two fish with idential markings were selected from each location. The following sex-linked pigment patterns, many of which are new for X. maculatus and which will be described in a forthcoming paper, were present in the fish from the Belize River and Sibun River. Macromelanophore pattern: A — Nigra: irregular black blotches along flank. < cc D Q Z o X CQ > 5 N tu CQ Q Z < oi > c2 z o S g s O cc Urn O o Ci. o Cx o c -s: a. a. 00 ON (N m (N ^ ly-j Tt 1— ' CO ^ ^ llrt ^ ^ (vj T-( rf (N 00 < CCCCCGC*^ OOOOOOO^ cccccGCri CO < CO < u s o Q- CO < u CO U >> Q < fN O ^ CN X o w u. o z o h H H CD z o u ui s o CD o S o U X u CO c H ■5 ^ S' Ci. o c Oo ONO\OOnONO<0\On ^ ^ ^ + 5* i ^ ^ u i A C^OnOOOO^^ ononoooooo 00 00ONONOnO\ON< X 4- + + +ft + Q Q xxxxxxxx rj fsi fS K K K X .5“ -S’ X Co 3 PU X" 5Q C) XX o ^ r- o ON C\ * + XX Q + XX ^ (N rYsr.lr 1936 18 Sp 20 Sr Ar^ 1900-9 'W + YavSp 1900-14J YvrYsr 1931 7 + 18 Br 1900-23 W+YavSp Jp XorYsr 1930 22 Sr 17 Dr 13 Dr 1 1900-6 W + Ycpo^r Hp-2 X + Ysd 1929 34 + 35 Sd 1900-4 W + YcP».r unknown 1909 53 + 1900-25 W + Ycpo.v Jp XorYsr 1972 10 Dr 14 Sr 1900-3 W + YsrSA unknown 1908a 15 + 1 1 Dr* 1900-3 Jp XspYsr 1908b 19 Sp 18 Sr 1900-7 W + Y JpAy 1900-11$ Y + Ya/fSd 1934 9 + lOMrSd 1900-21 X + X.4r Hp-2 X + Ysa 1923 19 + 10 Ar tThe sex chromosomes listed for the 15 females are the only ones that will adequately explain the results. tSex chromosomes of 1900-11 and 1900-14 are identified in Table 1. *Some pattern inherited from unknown male parent. ‘ Anal red, Ar, pattern of 1900-10 and of the 35 male offspring is different from that present in the 20 Sr females. ^ One male of each class developed some orange coloration in the iris. 1970] Kallman: Sex Determination and the Restriction of Sex-linked Pigment Patterns 7 ^henotype of offspring $$ 7 Ay* 10 + 9Sp 30 Sd 1 Ay Sd 15 Ay 0 Ay Dr* 16 Ay 1 ly Ay* 6Ir5p 1 1 Ir Sr 1 IrM' 3 Vo Sp Sd 28 Sd 7 Sp Sd= 1 Ay Sp CPo® 1 ly Ay Sp 5 Ay Sp Sd* 28 Ir Ay Sp 14 ly Ay= 19 Ay Sp 5 ly Ay Mr Sd* 1 + 2 ly Ay Sd 12 ly Ay 9 ly Ay Sp^ 19Iy Ay Sr^ henotype of offspring $$ 5 CPo 2 CPo N* 7 CPo Sp 7 CPo Sr 1 Sr 3 Sr 12 Sd Sr 2NSd 9SpN DNSd 9N 3Ay MrSd* 9 Ay 5 Ay Sd 5 Ay Sp 5 ly 14 Ir* S .Sp Ar' 19 Sr Ar' 5 Ay Sp Dr 12 Br Ay Sp 4 Ay Sp Sr 1 3 Dr Ay Sp 5 Sd CPo Sr 25 CPo Sr 3 CPo Sr 2 CPo N Sr 20 CPo N Dr 3 Br Sd 12BrSd Dr* 3 Br Sd Sr lOBrSd Sp 9 ly Ay^ 7 ly Ay Mr 5 7 Ar Sd 20 Sd A female of ped. 1923 with Ar was crossed to a Jp male. All female offspring but none of the male offspring (ped. 2050) inherited Sp, while Sr was inherited by males only. Therefore the genotype of 1923-1 must have been XX, one X derived from Hp-2, and the other, marked by Ar, from 1900-21. 1923-1 X+X.1, X JpXspY.,,s, Ped. 2050: 18 13 SpAr99', 33 ArSr$$ In ped. 2050 the Ar progeny fell into two non- overlapping classes. The 13 Ar females and 19 of the Ar males exhibited a red pattern quite unlike that present in Jp fish with Ar or in Jp x Belize hybrids that inherited the Ar factor of Jamapa. The unknown male which had already ferti- lized female 1900-2 (Table 3) at the time of capture, must have possessed the XY genotype. A wild-type female offspring (1905-2) was crossed to 1904-11, a Y^poY^y male. They pro- duced young of four classes (ped. 2124) : 7 N $9, 8+ $$, 3 N 33 and 2 CPo $$. The eight wild-type females presumably carry the CPo allele which is poorly expressed in many females. When a N male (2124-12) was bred with a Jp X£,,.Xpr female, all nine Dr offspring were females, all 13 Dr N were males (ped. 2245). Similarly a male with the CPo pattern (2124-11) sired 1 5 Dr females and 30 Dr CPo males (ped. 2234), when mated with a Jp female. These results indi- cate that CPo and N are T-linked (already con- firmed for CPo by ped. 1904 b. Table 3). The other sex chromosome of males 2124-1 1 and -12 must have been an unmarked X, traceable to the unknown male that fertilized 1900-2. The genotype of the wild-type female, 1905-2, was WX. Among the pedigrees listed in Tables 2, 3, and 4 are several exceptions that require further explanation. If the genotype of female 1900-2 were W only males of pedigree 1905 should have inherited the iris pattern (Table 3). However, there were seven female young with iris coloration and seven male offspring without any. Pedigree 2084 (Table 6) demonstrates that ly is Y-linked in females of ped. 1905. The females must have inherited the iris pattern from one of the unknown males that had inseminated female 1900-2 before she was collected. One of the males (1905-11, Table 5) with wild-type iris coloration was tested. He sired 32 Mr males and 29 ly males: the ly allele remained un- expressed in this male parent. However, it can- not be assumed that the other exceptional males (ped. 1905) were also due to nonexpression. The two males of peds. 1906a and 1907b (Table 6 Kallman: Sex Determination and the Restriction of Sex-linked Pigment Patterns 7 Zoologica: New York Zoological Society [55: 1 1970] Table 2. Inheritance of Pigment Patterns among the Offspring of Ten Females (ped. I89; Collected from the Sibun River, British Honduras (Freetown). Pedigree and genotype of parents 99 55 Pedigree of offspring 99 phenotype of offspring 55 1899-3 W+Y + t unknown 1916 8 Ay* 7 + 10 + 1899-5 W + Y + Gp XspYsd 1933 23 Sp 18 Sd ' 19 Sp 30 Sd 1899-7 W+Y.4, Hp-2 X + Ysf 1932 16 Sd 19 + 11 AySd 15 Ay 1899-2 W+?4V unknown 1913 8 Dr* 19 + 10 Ay Dr* 16 Ay 1 ly Ay* 1899-9 W + Yi. Jp X„Ysr 1981 24 Sp 14 Sr 16 Iri’p 1 1 Ir Sr 1 IrM’ 1899-4 W + Ys. 1900-13$ Y + Yr.«p 1927 17 Sp* 16 + 28 Sd 7 Sp Sd* 1899-6 unknown 1937 11 CPo* 11 Sd* 1 111 AySp CPo° 5 Ay Sp Sd* 19 Ay Sp 1899-10 W + Yj.s, 1899-12t Y„Y„ 1968 23 Ir 11 ly 11 lyAySp 28 Ir Ay Sp 1899-1 W + Y,,,.. unknown 1906a 7 MrSd* 9 4- 5Iy AyMrSd* 14 ly Ay* 1 + 1899-1 Hp-2 X+Ysj 1906b 12 Sd 9 + 12IyAySd 1 2 ly Ay 1899-8 W + Y,.,., Jp XspYs, 1928 17 Sp 11 Sr 19IyAy Sp’ 19IyAy Sr’ tThe sex chromosomes listed for the ten females are the only ones that will adequately explain the results. JSex chromosomes of 1899-12 and 1900-13 are identified in Table 1. •Some patterns inherited from unknown male parent. * An exceptional offspring with a new macromelanophore pattern linked to Ar on Y chromosome (Kallman ud * Vo is not expressed in females; 7 males were sacrificed at the age of 5 months before the pattern was apparent, ^Some fish also exhibit some red coloration in iris. Table 3. Inheritance of Pigment Patterns among the Offspring of Fifteen Females (ped. Collected from the Belize River. British Honduras (Bermudian Landing) Pedigree and genotype of parents 95 55 Pedigree of offspring 92 1 Phenotype of offspring 55 1900-1 W + Ycp. unknown 1904a 14 + 9 N* 5 Sd'l 16 CPo 2CPoN* 1900-1 Jp Xs.Yar 1904b 11 Sp 9 Sr i 7CPoSp 7 CPo Sr 1900-5 W + Y.r unknown 1911a 13 4- 1 Sd Mr* 11 Sr 1900-5 Hp-2 X+Y.J 1911b 21 4- 18 Sd ' 13 Sr 12SdSr 1900-22 W + Y.V Gp XspYsd 1952 24 Sp 3 Sd 1: INSd 9SpN 13 Sd 1900-24 W + Y.V Hp-2 X + Y« 1953 20 Sp 15 Sd 1( )NSd 9N 1900-8 W + Y.4V unknown 1917a 9 + lOMrSd* 1( )Ay MrSd* 9 Ay 1900-8 Gp Xs.Ysd 1917b 1 Sp 10 Sd i Ay Sd 5 Ay Sp 1900-2 W + Y,„ unknown 1905 29 + 1 Ir* 1 Mr" K )Iy 14 Ir* 2 Mr* 5 + 1900-10 W + Yxr Jp Xo.Ys.dr 1936 18 Sp 20 Sr Ar’ 1( > Sp Ar’ 19Sr Ar’ 1900-9 W + Y«s. 1900-14$ Yp.Yj,. 1931 7 4- 18 Br > Ay Sp Dr 12 Br Ay Sp 1900-23 Jp XorY 8r 1930 22 Sr 17 Dr 13 Dr 1’ lAySpSr 1 3 Dr Ay Sp 1900-6 W + Ycp«. Hp-2 X + Yad 1929 34 -1- 35 Sd 2^ > Sd CPo Sr 25 CPo Sr 1900-4 W+YcP.Sr unknown 1909 53 + 5- 1 CPo Sr 1900-25 W + Ycpo.v Jp Xz^rYar 1972 10 Dr 14Sr 1 CPo N Sr 20 CPo N Dr 1900-3 W+Y«,sd unknown 1908a 15 + 1 1 Dr» 12 IBrSd 12BrSdDr* 1900-3 Jp Xa.Yar 1908b 19 Sp 18 Sr 13 IBrSd Sr lOBrSdSp 1900-7 W+Yftd. 1900-11$ Y,Y„,s, 1934 9 + lOMrSd s ' ly Ay’ 7 ly Ay MrSd* 1900-21 X + Xd, Hp-2 X + Ya, 1923 19 + 10 Ar Ar Sd 20 Sd ■1. tThe sex chromosomes listed for the 15 females are the only ones that will adequately explain the results JSex chromosomes of 1900-11 and 1900-14 are identified in Table 1. •Some pattern inherited from unknown male parent. ^ red, Ar , pattern of 1900-10 and of the 35 male offspring is different from that present in the 20 Sr femalf^^ One male of each class developed some orange coloration in the iris. A female of ped. 1923 with Ar was crossed to a Jp male. All female offspring but none of the male offspring (ped. 2050) inherited Sp, while Sr was inherited by males only. Therefore the genotype of 1923-1 must have been XX, one X derived from Hp-2, and the other, marked by Ar. from 1900-21. 1923 1 X JpXgpY^j.g,. Ped. 2050: 18 5p9e; 13 5/7/Ir$5; 33 ArSr^^ In ped. 2050 the Ar progeny fell into two non- overlapping classes. The 13 Ar females and 19 of the Ar males exhibited a red pattern quite unlike that present in Jp fish with Ar or in Jp x Belize hybrids that inherited the Ar factor of Jamapa. The unknown male which had already ferti- lized female 1900-2 (Table 3) at the time of capture, must have possessed the XY genotype. A wild-type female offspring (1905-2) was crossed to 1904-11, a male. They pro- duced young of four classes (ped. 2124) : 7N9$, 8+ 9$, 3 N 55 and 2 CPo 55. The eight wild-type females presumably carry the CPo allele which is poorly expressed in many females. When a N male (2124-12) was bred with a Jp female, all nine Dr offspring were females, all 13 Dr N were males (ped. 2245). Similarly a male with the CPo pattern (2124-11) sired 15 Dr females and 30 Dr CPo males (ped. 2234), when mated with a Jp female. These results indi- cate that CPo and N are T-linked (already con- firmed for CPo by ped. 1904 b. Table 3). The other sex chromosome of males 2124-11 and -12 must have been an unmarked X, traceable to the unknown male that fertilized 1900-2. The genotype of the wild-type female, 1905-2, was WX. Among the pedigrees listed in Tables 2, 3, and 4 are several exceptions that require further explanation. If the genotype of female 1900-2 were only males of pedigree 1905 should have inherited the iris pattern (Table 3). However, there were seven female young with iris coloration and seven male offspring without any. Pedigree 2084 (Table 6) demonstrates that ly is Y-linked in females of ped. 1905. The females must have inherited the iris pattern from one of the unknown males that had inseminated female 1900-2 before she was collected. One of the males (1905-11, Table 5) with wild-type iris coloration was tested. He sired 32 Mr males and 29 ly males: the ly allele remained un- expressed in this male parent. However, it can- not be assumed that the other exceptional males (ped. 1905) were also due to nonexpression. The two males of peds. 1906a and 1907b (Table 8 Zoologica: New York Zoological Society [55: 1 2 and Table 4) lacking maternal pigment pat- terns were genetic sex reversals of the WY genotype (Table 6). This is evinced by the in- heritance of CPo and Sr in all male and some of the female offspring of ped. 2146 and by the appearance of Sr females (WX) and SdSr males (XY) in ped. 2069. Most probably the Sd males of ped. 1952 (Table 3) were genetic sex reversals of the WY genotype. None were tested, because an earlier analysis of a similar situation had shown that WY males often give rise to many more sex reversals (Kallman 1968). Such results would not help to identify the sex chromosome consti- tution of female 1900-22. Instead, one fish each of the “normal’ classes was tested (Table 6). Ped. 2065 demonstrates that the N gene of female 1900-22 was T-linked; ped. 2071 and 2090 show that her unmarked sex chromosome was a W. The eleven Sr and two /y males of the last two pedigrees are also sex reversals. Thus fish of ped. 1952 were of the following genotypes: Sp 99 — WX; Sd SS and 99 — WY, SpN XY; SdN $$ - YY. The occurrence of males with the exceptional genotype WY is not new for X. maculatus and one special case was analyzed recently (Kallman 1968). As in the previous study WY males arose not only among hybrids between two stocks, but were also found among the progeny when normal female sibs of sex reversals were mated to males of different, totally unrelated stocks (in these crosses to Jp and Up, peds. 2071 and 2090). Since the WY males of ped. 1952 {Bp 9xGp S), ped. 2071 [{Bp 9 x Gp S) <2 \ Jp 5] and ped. 2090 [{Bp 9 x Gp $) 9x Up-2 obviously have different genotypes, relatively few factors, perhaps only one or two with in- complete penetrance, must cause WY fish to differentiate into functional males. In contrast to the descendants of Np x Cp crosses (Kallman 1968), no WX males were herein encountered. Discussion Breeding data involving fish collected in British Honduras have demonstrated that the majority of females are WY and males YY. How- ever, one XX female and one XY male from the Belize River have been identified at Bermudian Landing and one XY male at Gaboural Creek. Therefore, the theory that the X chromosome is absent from X. maculatus populations in- habiting rivers in British Honduras can no longer be maintained. The X chromosome has been demonstrated throughout the range of X. maculatus, from the Rio Jamapa (Veracruz, Mexico) in the west to the Belize River in the east. There are a few locations where the X chromosome has not yet been found. No fish were examined from the Rio Tonala, Mexico. Only one female and two males were tested from the New River in British Honduras (Kall- man 1965). Information is also lacking for several small populations of platyfish in the streams and creeks of the narrow coastal plain of British Honduras, south of the Sibun River, but these populations inhabit an area that com- prises less than one per cent of the total range of this species. The results of the crosses herein described suggest the hypothesis that the W chromosome of natural populations does not carry pigment factors. This is unexpected because crossing over of pigment genes from the Y to the W has been reported in domesticated stocks of platyfish (Bellamy and Queal, 1951; Fraser and Gordon 1929; Gordon 1937) and in laboratory stocks derived from wild populations (Kallman 1965). The 27 marked females collected in the Sibun and Belize Rivers (Tables 2, 3, and 4) possessed a total of 41 patterns controlled by sex-linked factors (4 99 — macromelanophores only; 11 99— macromelanophores and red and yellow body patterns; 7 99 — red or yellow body patterns only; 2 99 — iris patterns only; 3 99 — red or yellow body and iris patterns) . Of the 41 factors representing at least three loci, not one was IF-linked. Since the females were obtained from three stations only, the sample may actually be smaller than it appears: several females of a collection could have been related and could have inherited their pigment genes from the same parent. Thus certain marked chromosomes would be represented more than once in the sample. This is probably true of the Sp^ and Vo alleles. The combination SpWo was present in two YY males from Bermudian Landing. No other sexually mature fish with Sp^ or Fo were collected®. However, even if each pattern or combination of patterns of each location is counted only once, the three collections com- bined are still comprised of 19 differently marked females with 28 pigment factors. The absence of IF-linked patterns, therefore, does not appear to be a sampling error. Certain red patterns develop poorly in females. Preliminary experiments have shown them to be under androgenic control. There exists the possibility that females might possess pigment factors on the W that would go undetected for many generations. It is difficult to surmise the possible function of a pigment factor that would be inherited strictly maternally but which could only be expressed in males. The possibility of “An immature fish with Sp* was present in the Gabourel Creek collection. It was too young to have any Vo exhibited. Table 4. Inheritance of Pigment Patterns among the Offspring of Five Females (ped. 1901) Collected from the Belize River, British Honduras (Gabourel Creek) 1970] Kallman: Sex Determination and the Restriction of Sex-linked Pigment Patterns 9 o Ci. o c a, c/2 < C/2 CO HI * ^ O •"I U U ^ ^ ^ ^ ^ i_, C'h ■<•<<•< CO CO U O vs m Tf O VO >o T3 -a i CO CO S „ * o O I- L >.CL| CU, CO Z < P c o (j o & a?- < CO <, CO CO CO U + a Cl. CO !0 1. Oo ■5 ^1: o Z CO Q CO CO CO ^ X) 03 JD ^ r-- 00 00 rj- o O o ^ ^ ^ (N 0\ o\ o\ o\ 0\ 0\ 0\ > + X ^ oa OQ . X X ^ + 5 § i xxxx cx O C Xt Oo c Cic “5 c c o o C (N C (N §p §p a cuO\ CO 00 03 &. a, a, fl, a, . t> t) >H + + + + + ''f Tt (5 Ui N n U m 9 crt Z Qj g G >>G ^ ic Z £0 2 V CO ^ o ^ ai <4-1 lx O (A bH (X c >-1 *5 5 n U etc H, O H Ji X « a E UJ T3 ^ 9 o ^ z ^ O o P 2 3 V3 H U H a z 2 O U i> a .i: S o o « CD W S "G sl 5x w c X > o 2 z ^ 2 ^ (-. > CO t3 aT3 CO >, cx a CO CO M CO CO ro vs On OV O O .-H -H (N r-l .-I CO ’13 °'2 0^ is uS Q O O.CU CO (j >> o. .i3 i (Z1 CO CO S a^J? a •< > •< S to M P CO OfnvofS^»oo>r^ ri (N m ^ (N (N •X 6c ^ -S .60 H-.. C "5 & X cu O (U6>6)6)6)D ac^aaacaa oooooooo CCCCCCCG ’-^^OO-H^OO I xr-oo»oa\rt**^0 »-4(NXOOrnrorN C^0^O^O^C^'^O^O^ • r- o\ o -N o o o o rt et rt (Ni rJ (N fi. T + *a fi. w 03 CO CO X ^ ^ X >H m cd T VO r-* r4 O O cu OV ON »-i W “ ^ ^ cc ^^X X + + >- O. -I- + * ^ VO rj VO On rj fN I O IT) «0 cu CU'^ ^ I *From 1927 $ (Table 2) x 1909 $ (Table 3). Frequency of Macromelanophore Patterns (M) in Collections of Platyfish from the Belize River and Sibun River (British Honduras) G-\ 1949% G 1950 G 1951 G 1952 G 1966 1966 Fi 1966 1970] Kallman: Sex Determination and the Restriction of Sex-linked Pigment Patterns 11 tu CQ < H s: § I a O, I ro o^ rj- r-i 'sO ^ r4 lo m Tj- 0\ r- (N O A o A o o A Tj- ^ 00 O 00 O V 2 CO ,N cO C -O ^ u S x: o o XI “ u !■§ o ^ A Co r- J3 .X Ov ^ vT Co T3 ^ C ^ cO cO . d c/3 1-1 c/3 ^ 3 i— 'as o T3 9 o "E O O B T3 in E c « •- c l| *o ^ tn lx ^ o a tn lx w o >. s £ « Vt (U ^ u ^ o « CO-- cO • O a « a o « a y CO E X> X CO ■£ S ' T3 •J O a X w o> 3 _C XJ c/3 §XS d'Hic Ov O s;o => T O ^ 00 V5 00 O c ® ^ d d .2 o ^ ® o ^ a, A B p XfL *d i> ll 5 “ E II C 3 r, C "S sp 2 o o •£ ■p o ° -o « C c ^ in ^ o 4> ^ *1^ ^ AT VJ Z o Q. 5 ID CO z o o z llJ (S> > X o 4 0 0 3 5 0 2 5 0 2 0 0 I 5 0 I 00 5 0 0 Python curtus WEIGHTS (kg) A-1.606 D-0. 07956 B-0.61478 E-0. 46009 C-0.06981 F-3.680 24 25 26 27 2 8 29 30 31 32 33 TEMPERATURE (“O Text-fig. 2. Oxygen consumption of Python curtus at various ambient temperatures. Circles, means; vertical lines, ranges. 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 23 produced the equation, C = 0.02W^-^“. The two equations are not significantly different. The re- gression coefficient is not significantly different from 1.0, indicating that the metabolism is di- rectly proportional to weight and not to the 0.67 power of the weight. We did not have sufficient data for animals of different weights at 21°C for direct compari- son with the above data. Therefore, a regression of metabolism on weight was calculated for data from ten boids of three species (Python curt us, P. m. molurus, P. m. bivittatus, and P. reticula- tus) at 26.0°C to 28.4°C. These calculations resulted in the equation, C = 1.975W®-®®‘‘ where metabolism is, indeed, proportional to weight to the 0.67 power. For a comparison with Bene- dict’s data, values for ten boids at 27.1°C to 29.1°C were taken from his work. These include eight of the animals and all four species used by Galvao et al. (1965) to calculate the regres- sion at 19.5°C to 23.3°C The equation obtained was, C = 0.395W® *®2 xhe exponent is not sig- nificantly different from 0.664 figure obtained in this study or from the values of 1.09 and 1.12 calculated by Galvao for his data at 21.5°C or for Benedict’s data at 19.5'’C to 23.3°C. All four regression equations discussed above and the individual values from this study are shown in text-fig. 5. The relation between metabolism and weight depends on the temperature at which the metabo- lism is measured. It is also likely that a better comparison could be made, if the data were calculated for single species, rather than lumping together data from several species of two sub- families (Pythoninae and Boinae). Baldwin (1930) gave oxygen consumption data at 20°C for 13 specimens of Pituophis sayi weighing 272 grams to 835 grams. No data were given on the acclimation conditions. By con- verting the oxygen data to calories per hour and calculating a regression of heat production on weight, we obtained the equation, C = 8.96 1W°-^®®. The data are highly variable, the 95 percent confidence limits on b being ± 0.797. A “t” test on b (S,, = 0.362) was not significant TEMPERATURE (“C) Text-fig. 3. Oxygen consumption of Python molurus at various ambient temperatures. Circles, means; vertical lines, ranges. 24 Zoologica: New York Zoological Society [55: 2 (P<0.20). Unfortunately, Baldwin provided no information that gives any clue to the source of the variability of the data. Heart Rates and Oxygen Pulse Difficulty was encountered in obtaining heart rates from pythons. The time interval necessary for the animals to settle down after having the EKG leads placed on them also provided suf- ficient time for them to work the leads off. In spite of this problem heart rates were obtained for Python molurus acclimated to several tem- peratures. Rates varied from about three per minute at 17°C to about five per minute at 26.5°C. At 33°C the rate had increased to about 16 per minute (text-fig. 6). The response of heart rate to temperature apparently parallels the increase of oxygen uptake with temperature. Oxygen uptake increases from about 5 cc kg-^ hr-^ at 15.5°C to 12 cc kg-^ hr-^ at 27°C. The rate of increase then rises until the uptake is about 30 cc kg-^ hr-^ at 33 °C (text-fig. 3). This correlation is shown more readily when the data are calculated as oxygen pulse (Table 1 ) ; with increasing temperature the heart rate in- creases proportionately with oxygen consump- tion and oxygen pulse remained fairly constant. Jayasinghe and Fernando (1964) found a rate of 40 beats per minute for Python molurus. They failed to report either the acclimation or the measurement temperatures. The size of the animals was given as between 10 feet and 20 feet (three meters to six meters). Animals of these lengths could weigh between 10^ grams and 10® grams. Therefore, without specific tem- perature and weight information, no direct com- parison can be made with these data. Heart rates of a 4313 gram, male boa con- strictor, Constrictor [ = Boa] c. constrictor y/ere given by Clarke and Marx (1960). The snake was kept at 23°C to 27°C for some time before the measurements were made. Heart rates were taken over a four-hour period during which the temperature was dropped from 23°C to 18°C and the heart rate dropped from 15 beats to 12 beats per minute. Text-fig. 4. Oxygen consumption of Python reticulatus at various ambient temperatures. Circles, means; vertical lines, ranges. 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 25 Table 1. Heart Rate and Oxygen Pulse Data for Python molunis. Temp. ro C>2 Cons, (cc kg-^ hr-^) Heart Rate (beats/ hour) O2 Pulse (ccOi beat'^ kg"^) Snake Wt. (kg) 16.9 5.86 198 2.96 X 10-2 17.08 20.8 6.40 240 2.67 X 10-2 29.41 20.9 6.58 300 2.19 X 10-2 21.68 26.5 8.37 300 2.79 X 10-2 22.94 32.8 27.07 960 2.82 X 10-2 21.68 Rebach (1969) measured heart rates of boa constrictors weighing from 0.393 kilograms to 8.51 kilograms at temperatures of 20.0°C and 32.2°C. Rates at 20.0°C were 6.4 ( 5. 0-8.0) beats per minute and at 32.2°C were 23.9 ( 13.0-30.0) beats per minute. When the smaller size of these snakes is considered, the measured heart rates are in good agreement with those of the pythons measured in the present study. Mullen (1967) measured heart rates of sev- eral temperate zone lizards and snakes, all of which are of small size compared to pythons. Mean heart rates for the snakes varied from about 43 beats per minute at 22°C to about 95 beats per minute at 30°C. These rates are con- siderably higher than the rates found for the Indian python but the greater mass of the python probably accounts for its lower heart rates. Bartholomew and Tucker (1964) demonstrated an inverse correlation between heart rate and weight in varanid lizards; this correlation also occurs in other animals. WEIGHT ( g ) Text-fig. 5. Correlation of heat production with weight of boids based on the data of several investigators. Regression equations obtained by the method of least squares are as follows: Benedict ( 19.5-23.3), C = 0.02W112; Benedict (27.1-29.1), C = 0.395W0-852; Galvao (21.5), C = 0.04Wi <>9; Vinegar (26.0-28.4), C = 1.975Wo ®®^. Individual points are plotted for data from this study only. 26 Zoologica: New York Zoological Society [55: 2 Text-fig. 6. Heart rates of Python molurus at several ambient temperatures. Weights and sample size for each point are shown in the upper left of the figure. Circles, means; vertical lines, ranges. Metabolic Responses to Temperature Change Specimens of Python molurus bivittatus (NYZP No. 630514) and P. reticulatus (NYZP No. 640670), 17.08 kilograms and 25.08 kilo- grams, respectively, were subjected to rapid changes in temperature of about 15°C. The ani- mals were initially acclimated to the lower temperature for a two-week period. Temperature was then increased, kept at the high setting for nine days and finally returned to the low setting. Gas exchange was measured over the whole period (text-figs. 7 and 8). The metabolic re- sponse to temperature rise was slightly different in each of the animals. Python molurus showed an initial increase from about 5 cc kg-^ hr-^ to 40 cc kg-i hi^i within two days and then a gradual drop to 20 cc kg-^ hi^^ within five days. Python reticulatus showed the same initial in- crease but remained at a level of about 35-40 cc kg-i hr-i for the nine day period. Decreased temperature seemed to produce a slight meta- bolic undershoot in P. molurus, while P. reticu- latus returned immediately to its initial level. Further measurements are necessary before any conclusions can be reached regarding the con- sistency of these responses. Brooding Metabolism of Python molurus bivittatus An Indian python (Python molurus bivittatus, NYZP No. 630514), 14.25 kilograms in weight and 2.7 meters in length, laid 23 infertile eggs on or about February 15, 1965. The animal was found coiled around the eggs on February 18 and was transferred, without disturbing her or the eggs, to a respiration chamber in a tempera- ture controlled (±; 1°C) room. Measurements of gas exchange, temperatures, and contraction rates were made for the 30-day period that the python remained on the eggs. Additional gas exchange measurements were taken 40 days after the end of the brooding period to obtain non-brooding values. Oxygen consumption cal- culations were based on the weight of the snake at 14.25 kilograms during brooding and at 12.37 kilograms during non-brooding. 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation, During Brooding of the Genus Python (Reptilia, Boidae) 27 I OCTOBER 1965 NOVEMBER 1965 Text-fig. 7. Metabolic response to temperature change in a 17.08 kilogram Python molurus bivittatus. Temperature changed from 15.4° to 31.7°C on October 27 and back to 15.4°C on November 5 (arrows). Oxygen consumption during non-brooding was typical of an ectothermic animal, decreas- ing with decreasing temperature (text-fig. 9, lower curve); but oxygen consumption during brooding was similar to that of endothermic animals (text-fig. 9, upper curve). At about 33°C, the metabolic rate of the python is ap- proximately the same during brooding and non- brooding. However, the oxygen consumption of the brooding animal increased when the tem- perature was decreased from 33°C to 25.5°C. Thus, 33°C appears to be analogous to the “lower critical temperature” of birds and mam- mals. The analogy is enhanced further by the onset of muscular contractions which accom- pany the increased metabolism at temperatures below 33°C. The frequency of contractions in- creases with decreasing temperature and increas- ing metabolism (text-fig. 10). A similar correla- tion exists between the temperature differential between animal and air and the contraction rate (text-fig. 11). A maximum temperature differ- ential of 4.7°C was maintained at an ambient temperature of 24.8°C (text-fig. 12). A full account of the data for this brooding was given by Hutchison, Dowling, and Vinegar (1966). On February 15, 1966, a specimen of Python molurus bivittatus (NYZP No. 630514) was found coiled in a corner of an exhibit cage. One egg, which was opened and found to be infertile was noted beside her. No other eggs were laid at the time. The animal was taken to the labora- tory and placed in a respiration chamber. She weighed 17.25 kilograms (including the ejected egg) . Twenty-one additional eggs were laid dur- ing the night of February 16-17, 1966. Respira- tion, temperature, and contraction rate data were collected as during the brooding period of the previous year. Several eggs were removed during the course of brooding as they started to turn yellow. On March 28, 1966, the remain- ing eggs, which were all infertile, were removed from the female as all signs of regular contrac- tions had stopped. Irregular contractions were noted until April 8, 1966. 28 Zoologica: New York Zoological Society [55: 2 2 5 2 6 2 7 2 8 2 9 3 0 3 I 1 2 3 4 5 6 7 8 9 OCTOBER 1965 N0VEMBER1965 Text-fig. 8. Metabolic response to temperature change in a 25.08 kilogram Python reticulatus. Tempera- ture changed from 15.5° to 31.5°Cbn October 27 and back to 15.5°C on November 5 (arrows). Although tidal volume could not be deter- mined quantitatively, qualitative observations were made. The number of inspirations per minute increased with increasing contraction rate. Contraction and breathing rates were: 2, 2; 9, 4; 33, 6; 37, 6. The normal inspiration rate in a large python is about two per minute. Al- though the frequency of inspirations did not increase greatly with increasing contractions, the tidal volume did increase. The breaths of six per minute were quite noticeably deeper than those at two to four per minute. Measurements were also made of the height and basal width of the snake’s coil at different contraction rates. Calculations of volume and area were made assuming the coil to be a solid cone. A brooding P. m. bivittatus is pictured in Plate I. Table 2 shows the calculated data; the surface area calcu- lated includes that part in contact with the sub- strate. Surface area to volume ratios decreased with increasing contraction rates. The metabolic data were similar to those obtained in the pre- vious year. Text-fig. 13 shows the oxygen con- sumption-contraction rate data for the two years. Data for contraction rates at various temperature differentials are plotted in text-fig. 14, and body temperature-ambient temperature data are shown in text-fig. 15. During most of the 1966 brooding, a 17.1 kilogram specimen of P. m. molunis (NYZP No. 640578), was kept in a second respiration chamber. Its metabolic re- sponses to temperature were also determined. Data for the two animals are shown for an 11 -day period at various temperatures in text- fig. 16. False Brooding Behavior in a Female Python molunis moliirus On May 31, 1966, a specimen of Python molunis molunis (NYZP No. 640578) was seen contracting at an uneven rate in a manner similar to a brooding python. Since contractions of this type had been seen previous to the last two egg layings of P. m. bivittatus (NYZP No. 630514), it was assumed that No. 640578 would probably lay eggs within the following few days. The ambient temperature of the cage and surround- ings of the python was about 27°C. It was trans- ferred to the laboratory (at 31°C) and placed in one of the metabolism chambers so that she 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 29 Text-fig. 9. Oxygen consumption of a Python molurus bivittatus at different ambient tempera- tures. Upper curve: animal during brooding. Lower curve: same animal during non-brooding. Circles, means; vertical lines, range, (from Hutchison, V. H., H. G. Dowling and A. Vinegar, 1966). Text-fig. 11. Correlation of contraction rate with temperature differential in a brooding Python molurus bivittatus. Line and regression equation calculated by method of least squares. Circles represent individual measurements (from Hutchi- son, V. H., H. G. Dowling and A. Vinegar, 1966). Text-fig. 10. Correlation of rate of body contrac- tions with oxygen consumption in a brooding Python molurus bivittatus. Dashed line and regres- sion equation calculated by method of least squares. Circles, means; vertical lines, range of oxygen con- sumption; horizontal lines, range of contraction rate (from Hutchison, V. H., H. G. Dowling and A. Vinegar, 1966). Text-fig. 12. Correlation of body temperature of a brooding Python molurus bivittatus with ambient temperature. Dashed line indicates equal ambient and animal temperatures. Circles, means; vertical lines, range of animal temperature; horizontal lines, range of ambient temperature (from Hutchison, V. H., H. G. Dowling and A. Vinegar, 1966). 30 Zoologica: New York Zoological Society [55: 2 Table 2. Coil Size Data for a Brooding Indian Python. Tb-Ta (°C) Contraction Rate Per Minute Coil Ht. (cm) Coil Width (cm) Coil Vol. (cm^) Coil Area ( cm^) Area/ Vol. 1.6 2-3 11 50 7198.13 4106.34 0.57 3.4 30 16 48 9649.15 3982.02 0.41 4.8 42-45 20 46 11077.26 3862.33 0.35 could lay her eggs there. Gas exchange measure- ments were made from June 13 to June 16 and July 2 to July 12, 1966. Ambient temperature was changed several times during these periods. No eggs were laid and the animal was removed from the laboratory on July 12, when muscular contractions were no longer observed. Occa- sional irregular contractions were noted until September 2. Offerings of food were consistently ignored. On October 6 the python was force fed one rat. A mucous discharge was seen com- ing from the mouth on October 25. The animal seemed to be having trouble breathing and was euthanized with nembutal on November 1. Au- topsy confirmed that the animal was a female. The contractions of the animal were more regular and the response to temperature change more pronounced from June 13 to June 16 than from July 2 to July 12 (text-fig. 17). A decrease in ambient temperature from 31°C to 26°C on June 14 resulted in an immediate increase in CONTRACTIONS / MINUTE Text-fig. 13. Correlation of rate of body contrac- tions with oxygen consumption in a brooding Python molurus bivittatus. Regression line repre- sents data from 1965 shown in Fig. 10. Circles represent data from the same individual for 1966. metabolic rate. On July 5, a similar decrease from 32° to 25.5°C resulted in an initial de- crease and then in an increase in metabolism, although not to the level reached on the earlier date. By this time the metabolic response to tem- perature change was slight. These observations and the irregular contractions seen in P. m. bivit- tatus (No. 630514) prior to egg laying suggest that physiological thermoregulation in pythons is under hormonal control. The irregular con- tractions in No. 630514 up to one week prior to egg laying suggest an increase of certain hormone levels to that required for thermo- regulation. The sluggish response to temperature change in No. 640578 probably reflects a change in hormone level towards the normal non-brood- ing condition. This, however, does not explain the brooding behavior and thermoregulation in an animal that has not laid eggs. A possible explanation might be a malignancy affecting the brain area controlling the whole brooding re- sponse or the gland responsible for secreting a hormone involved in brooding. Since the normal brooding period is from one-and-a-half to two- months long, and since this animal continued to show irregular brooding behavior for over three months, further support is given to the malig- nancy hypothesis. Brooding in Various Python Species Python curtus Noble (1935) described the laying of 16 in- fertile eggs and the brooding of a blood python. Python curtus. Temperatures reported were taken with a gas-filled mercury thermometer. All of the body temperatures reported were intermediate between the temperatures of the substrate and the air, and indicated that the snake had not developed an elevated body tem- perature. However, the substrate temperatures reported were 31.5°C, 31.8°C, and 32.2°C. These temperatures are in the vicinity of the lower critical temperature (33°C) found in the present study for P. molurus, therefore the “thermostat” of Noble’s python may not have been “calling for heat.” No conclusions regard- ing the ability or inability of P. curtus to thermo- regulate can be drawn from Noble’s obser- vations. 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 31 o o UJ tr UJ u. u. Q UJ a: 3 I- < oc UJ Q. UJ Text-fig. 14. Correlation of contraction rate with temperature differential in a brooding Python molurus bivittatus. Regression line represents data from 1965 shown in Fig. 11. Circles represent data from the same individual for 1966. Text-fig. 15. Correlation of body temperature of a brooding Python molurus bivittatus with ambient temperature. Dashed line indicates equal ambient and animal temperatures. Squares, data from 1965; circles, data from 1966. OXYGEN CONSUMPTION (cc 32 Zoologica: New York Zoological Society [55: 2 100 t\j o 50 — 1 1 1 1 T 1 1 1— T 1 1 — 1 • • BROODING r • • • • • ■ NON -BROODING • • •• . - • " - • _ • • - • - • • - •• • _ • • • • • •••? • • ■ ■ _ _ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ -■■■■■■■■■■■■ ■■■ - _J ^ ^ _i 1 1 1 1 J 1 . T . 12 13 14 15 MARCH 1966 Text-fig. 16. Oxygen consumption of brooding and non-brooding Python moluriis under identical ambient temperatures. Temperature changed from 24° to 31.5° on 10 March, to 30° on 13 March and to 23.5°C on 18 March (arrows). Additional definite information regarding the ability of P. curtus to thermoregulate physiologi- cally while brooding its eggs was obtained dur- ing the present studies. A female blood python laid fertile eggs on May 29, 1965, while none of the project personnel was available. The ani- mal was removed from her eggs at that time and would not return to them on May 31 when she was placed back with them. Nevertheless, on June 1 when she was placed in a room at about 27°C, she contracted her musculature at a rapid but irregular pace of about 27 contractions per minute. Irregular sporadic contractions had been noticed six weeks prior to egg laying. No further data were obtained from the python because normal brooding information could no longer be obtained. However, this evidence suggests a second species of python that has some ability to respond physiologically to decreases in am- bient temperature while brooding eggs. Chondropython viridis Evidence exists for a third species having the ability to control its temperature while brooding. Kratzer (1962) reported on mating and brood- ing of Chondropython viridis. After egg laying the female python showed muscular twitching at intervals of two to three seconds. The tem- perature of the substrate was reported at 28°C and that of the air, 26°C to 30°C. However, these temperatures were obtained a month or more before the eggs were laid. If the tempera- tures were about the same after laying, the observed contractions would be expected in an animal capable of such a response to tempera- ture decrease. Python reticidatus Little information exists regarding brooding in Python reticulatus. Wall (1926) described various aspects of the biology of the reticulated python. In his 'section on brooding he stated that “Experiments prove that the dam’s body temperature is not raised during this period.” However, he did not state whose experiments these were or under what conditions they were performed. Lederer (1944) made some observa- tions on a brooding animal of this species and 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 33 Text-fig. 17. Oxygen consumption of a Python molurus showing false brooding behavior. Temperature changed from 30.5° to 26° on 14 June, to 33° on 16 June, to 25° on 5 July and to 30°C on 8 July (arrows). found that the snake’s temperature was close to that of the substrate which was several degrees higher than the air temperature. Unfortunately, the lowest substrate temperature reported was 32.2°C, which is the approximate critical mini- mum temperature for the Indian python. The animal, therefore, probably was not observed under conditions that would have elicited the thermoregulatory response. Some information that may be more useful in determining whether P. reticulatus is capable of maintaining its temperature above that of the environment by physiological means was pro- vided by a visitor from Malaya to the New York Zoological Park. The information was given to Assistant Animal Manager Peter Brazaitis by the visitor, K. J. Sims. Sims had a 20-foot python that laid 67 eggs in an outdoor enclosure. Since Sims worked up to 14 hours a day, many of his observations, were made in the evening when the temperature was already cooler. Dur- ing all of his observations he never saw the ani- mal undergo muscular contractions. These ob- servations suggest that the reticulated python lacks any physiological thermoregulatory ability. On January 29, 1968, a P. reticulatus, weigh- ing 73.19 kilograms, laid 51 eggs at the New York Zoological Park. The animal died two days later so that little information was obtained. However, at no time did anyone see any signs of muscular contractions. No other reports were found of P. reticu- latus brooding in zoological gardens or in na- ture that would clarify the brooding situation in this species. The information that is available indicates that this species does not have the same thermoregulatory ability that has been demonstrated in P. molurus. Python sebae Brooding in Python sebae was reported by Sclater ( 1862). He made simultaneous tempera- ture measurements of a 14-foot male and a 22- foot brooding female python. The eggs were laid on January 13 and were removed from the fe- male on April 4. She had left the eggs only a few times during the whole brooding period. Temperatures between the coils of the male and female on each of four different days were: 34 Zoologica: New York Zoological Society [55: 2 74.8°F, 81.6°F; 74.0'’F, 83.2°F; 76.0°F, 96.0°F; 77.6‘’F and 86.0°F (23.8°C, 27.6°C; 23.4°C, 28.5°C; 24.5°C, 35.6°C; 25.4°C, and 30.0°C). Air temperatures during each of the previous measurements were: 58.6°F, 65.4°F, 60.0°F, and 66.0°F (14.8°C, 18.6°C, 15.6°C, and 18.9°C). No conclusions regarding the physio- logical thermoregulatory ability of P. sebae can be reached on the basis of these date. Although the temperatures of the male and female pythons were both above the air temperature, no mention of the floor temperature was made except that the cage was heated by hot water pipes. It seems likely that the animals were warmed by con- ductive heat from the water pipes ( probably during the night when the snakes are more ac- tive). The female would have retained heat better than the male because her larger size and coil around the eggs presented less surface area per unit mass for heat loss to the environment. Fitzsimons (1930), in referring to brooding of pythons, stated: “At this period her blood rises to a temperature of 90° Fahrenheit, which is apparently. Nature’s rule for the hatching of infant pythons.” The source of this information was not stated. It is apparently a generalization that Fitzsimons made as a result of reading some of the early accounts of python brooding temperatures, since he referred to these accounts without giving the specific sources. Benedict (1932) gave an account of respira- tion rates and temperatures of a brooding 4.6 meter specimen of P. sebae at the National Zoological Park in Washington, D.C. The meas- urements were made on one day only. Average respiration rates during the course of the day ranged from 2.0 to 3.1 breaths per minute. Three sets of measurements were made during the day. Temperatures of the gravel around the python ranged from 29.92°C to 32.82°C; of the air 10 centimeters to 15 centimeters above the floor, 29.20°C to 32.04°C; of the air 30 centi- meters above the floor, 29.30°C to 32.02°C; of the air 60 centimeters above the floor, 29.14°C to 31.44°C; under the python, 32.07°C to 34.62°C: between folds of python, 33.33°C to 35.18°C. During the three sets of measurements Benedict recorded temperature differences be- tween snake and environment of up to 3°C or 4°C. However, some information given by Benedict casts doubt on the validity of his con- clusions. The incubating python was located near a glass window that was a few centimeters from the air in the corridor outside the cage. The air outside the cage was reported as 20.4°C. In 1960, Dowling (I960 and unpublished data) observed two brooding female pythons. One was a 53.07-kilogram (including eggs) specimen of Python moluriis (NYZP No. 540616) and the other a 20.86 kilogram (includ- ing eggs) specimen of P. sebae. The former laid 53 eggs on April 6 and the latter laid 45 eggs on April 11. Temperatures of the animals, sub- strate, and air were taken at various times dur- ing the brooding period. Hourly readings for both animals were made from 0745 on April 29 until 0745 on April 30. The temperatures re- corded for the animals and the adjacent sub- strate are shown in text-fig. 18. Text-fig. 18A shows that the body temperature of the P. sebae followed that of the substrate when the substrate temperature was lowered. Text-fig. 18B shows that the body temperature of P. moliiriis while brooding is relatively independent of the sub- strate temperature. Dowling noted that the Indian python contracted its musculature at a rate that was inversely proportional to tempera- ture. No muscular contractions were seen in the African python. Text-fig. 19 shows the relation- ship between contraction rate and temperature differential (animal temperature minus substrate temperature). These data are presented for the above Indian python and for another individual (NYZP No. 510720) weighing 43.41 kilograms, including eggs laid on April 5, 1961. The Afri- can python was seen to leave its eggs during brooding and to go into the heated pool in the cage and later return to the eggs. It seems prob- able that the African rock python also lacks the thermoregulatory ability exhibited by the In- dian python. Morelia spilotes variegata Cogger and Holmes (1960) produced good evidence to show that the carpet python (Morelia spilotes) regulates its temperature behaviorally. The animal basks in the sun and then forms a tight coil while resting. Heat was retained even through the night. However, if the following day was cloudy, the animal eventually came into equilibrium with the surrounding air. The data would have been more convincing if substrate temperatures had been given. The warmest tem- perature recorded for the snake on a sunny day was about 90°F (32.2°C), which is the lower critical temperature demonstrated for brooding Indian pythons. Cogger and Holmes suggested that Morelia may regulate its temperature simi- larly while brooding its eggs. Egg Brooding in Various Reptiles Reptile eggs get various degrees of care by the parent after being laid. Many eggs are merely laid in a hole in the ground or under the bark of a fallen log and left to the elements. Some animals lay their eggs in places that do not fluctu- ate much in temperature or humidity. One such TEMPERATURE 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 35 15 10 15 20 25 HOURS Text-fig. 18. Body and substrate temperatures over a one day period for two brooding pythons (Python molurus and P. sebae). Key to symbols in lower right of figure. place is in termite nests. An account of tempera- ture regulation in termite nests was given by Luscher (1961). The South American teid, Tupinambis nigropiinctatiis, lays its eggs in termite nests (Hagmann, 1906). Gehyra pilbara, an Australian gecko, not only lays its eggs in mounds of the termite, Eutermes triodiae, but also lives in the mounds, thus escaping the rigors of the surrounding desert (Mitchell, 1965). A small python, Liasis childreni perthensis, which reaches an adult length of 30 centimeters, is found also in the nest with Gehyra. Although Mitchell did mention that Gehyra is a food item of the python, no information was given as to whether Liasis also lays its eggs in the termite mound. Noble and Mason (1933) summarized the literature up to that time on various snakes and lizards that actually stay with or brood their eggs. While some of the animals may sun them- selves between periods of brooding, their size makes it unlikely that they contribute any ap- preciable heat for the development of their eggs. Most cases presented seem to be examples of egg protection rather than egg incubation. Noble and Mason (1933) provided some of their own data on brooding in Eumeces fasciatiis, E. lati- ceps, and Ophisaiiriis ventralis. Body tempera- tures of Eumeces were reported to be 1.6°C to 3.2°C higher than that of the eggs. However, mention was made also of how quickly the body temperatures could change. This, along with the infrequency of temperature readings, casts doubt on any degree of thermoregulation existing for Eumeces. The authors concluded from similar infrequent data recordings that Ophisaurus prob- ably does not have any thermoregulatory ability. A more detailed account of brooding in Ophisaii- riis was given by Vinegar (1968). A detailed account of the manner in which the Nile monitor, Varanus niloticiis, makes use of the nests of the termite, Nasutitermes trinervi- formis, for incubation of its eggs in Natal, was given by Cowles (1930). Kopstein (1938) sum- marized several papers concerning the snake Boiga drapiezii and its laying of eggs in the nest of the termite, Lacessititermes batavus. Energetics of Python curtus No previous attempt has been made to con- sider a complete energy budget for a snake. With 36 Zoologica: New York Zoological Society [55: 2 Text-fig. 19. Correlation of body contractions with temperature differential (body temperature — substrate temperature) for two different Python molurus bivittatus. Lines and regression equations calculated by method of least squares. Key to symbols in upper left of figure. hope of accomplishing this, in August 1965 three hatchlings of Python curtus were placed in indi- vidual cages in a temperature-controlled room (27°C). The animals were kept at this tempera- ture except for periods of several weeks when they were acclimated to different temperatures for measurement of gas exchange. A water bowl was kept in each cage. Food consisted of albino mice except for one snake which was fed rats after 14 months. The food was weighed before being fed to the snakes. After each defecation the pythons were weighed. Lengths were not taken because of the difficulty in getting the ani- mals stretched out. The severe struggling of the animals would have injured them and probably made them stop feeding. The defecations and renal waste were frozen and later oven-dried, weighed, and the caloric content determined. Caloric content was also determined for mice and for a python (a young individual of P. setae). The caloric value obtained from this ani- mal was used to calculate the energy budget of Python curtus, but is a tentative value, to be replaced as soon as caloric determinations of a series of P. curtus can be made. Caloric values were determined with a Parr Adiabatic Oxygen Bomb Calorimeter. Six de- terminations were made on each sample with the mean value being used as the caloric value of the sample. Text-fig. 20 shows the growth of the three P. curtus over a period of two years (August 1965 to July 1967). A marked difference in growth rate is evident. Photographs taken on February 14, 1967, emphasize this difference (Plate II). Some explanation of this divergence in growth was sought and found partially in the behavioral history of the animals. Individual No. 650495 showed aggressive behavior from the date it hatched. It is the only one of the three pythons that has attempted to bite. All three ani- mals initially had to have food placed in their mouths to induce them to eat, but python No. 650495 was the first one to start feeding by itself. Number 650503, which showed signs of aggressiveness only when forcedly excited, was the second individual to start feeding by itself. Python No. 650496 could not be induced to bite and never took food voluntarily. WEIGHT (KILOGRAMS) 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 37 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.0 • 650495 • - ■ 650496 • A 650503 - PYTHON CURTUS • - • - - • - - • A A - • A A ■ A tk A • ■ ■ ■ ■ ■ 0 N D 1965 MAM J J A •1966 0 N A M 1967 Text-fig. 20. Growth of three hatchling Python curtus over a two-year period. Table 3 summarizes the feeding and growth data of the three pythons for their first two years of life. The data for No. 650496 show the very slow rate of growth for each weight period. A slight loss of weight occurred for two periods, indicating that the food consumed since the previous weighing was enough to sustain life but not enough for additional growth. Individual No. 650495 shows a much higher growth rate; the maximum for one period was 9.18 grams gained per day for a 74-day period compared with a maximum of 0.27 grams gained per day for a 65-day period for No. 650496. The rates of growth for No. 650503 are intermediate between the other two animals. Another factor contributing to the difference of size attained by these animals is shown in Table 3. Data are shown which express the amount of food con- sumed which goes into producing new python protoplasm. These data are expressed as weight gain divided by food consumed times 100. The values based on total weight gain and total food consumed are for No. 650495, 52 percent; No. 650503, 45 percent; No. 650496, 29 percent. Not only was No. 650496 not consuming as much food as its siblings, but the low value of 29 percent indicates it was not using food as efficiently as the others. Caloric values of the intestinal and renal wastes of the pythons are summarized in Table 4. Laboratory mice under 10 grams (wet) had a caloric content of 5204.39 ± 92.25 calories per gram (dry), while those over 10 grams (wet) had a caloric content of 5460.15 ± 48.34 calo- ries per gram (dry). The specimen of Python sebae weighing 173.69 grams (wet) had a con- tent of 4136.22 ± 67.51 calories per gram (dry). The ratio of dry weight to wet weight for the mice was 0.27, for the python, 0.21. Oxygen consumption of Python curtus is summarized in text-fig. 2. The caloric values, oxygen consump- tion data, and growth figures can be used to calculate an energy budget for the pythons over any growth interval for which there is complete data. Table 5 shows such an energy budget for each of the three pythons. Discussion of Reptile Energetics Pope (1965) gave an account of an Indian python. Python molurus, eating 123 laboratory rats (61 pounds) during its second year and most of its third year. The snake increased its Table 3. Food Consumption and Growth of Three Young Python curtiis Hatched from Same Clutch of Eggs. 38 Zoologica: New York Zoological Society [55: 2 1) E Ut (L> O ^ S o ON o cs »-H cn ^ r-- xj- o o NO OS o 00 r- 00 O r- 1-H VD (N VD VD ov ON 00 fO VD 00 On q q I— t q 00 00 ’-H q q q q IZN T-H VD VD d m m* d ON VD cn cn t-H d VD VD 1-H r-- VD m VD m (N 00 »-H VD ON (N ON \r\ m VD o r- 00 tT) (S (N cn »r) O (N cn o (N n >n VC 00 ^ I I I VC O 00 o vq rn ri (N n 00 (N n n lo o VO d d o OV -H 0 F' lo O >q ON O ^ >0 On ■'t 'T '-I d d VC 4- fS d 00 O "-H (N ro VC ri ri ^ ^ ^ n 00 o ^00 ON VC U- VC PJ rj ro T F' d 00 00 00 o vq >-H >o F~ d ^ F^ ON f4 VC F^ ON ^ F^ O') — I 00(OF^F^>00— I O ':!• Tj- ^ O VC >o ON 00 F~ 00 00 00 Fn) O m m ON fv| F^doNF^'Tvcdm I F^ F^ VC »— < 00 00 -H ^ rl r-< n ro m m n oJ FN ro F~ O O ON F^ n m FN F^ ^ £ • o S G c: o o ;g >F1 On ■T O IFN NO d Z "cS s E < ONrt00VOFn>— lOjF' OCoOF^OVOlFiONlq ^^oo^on^f^iO t^ONOooo^oorlF' '— i-^rnmFNOF'V-i NO ON o >FN VC O f-JOf^— I'-'io^vo 7 VFNNO'FN'TlFN'TVCn "a E 'E < «fnifnONF''TOOO qF^lq^^qqq did-H'^ONddd OvONOlFiF'ONVOiFl FJ Fl NO --I ^ IFl NO 'T O FN 00 NO m FN IFN d 00 F' 'Vf F^ On _ FI VO F4 'T ON FN g >Fl o VF) VO 'T FN OF! VC rn Tj- Fl -F IT) vC F~ qONTtioq-Tl- FN FI d F-^ d d -F FI I -F FI I d z 13 s 'S < ON ON IF) O FN ON IF) ^ F^ q q FI G On FN -F ON >F) FI ON F~ >F) O Ff FI FI '-V FN ^ O F-) F) >F) IF) IF) VC F-) NO ON On q q ff IF) q F IF) On -F vd IF) ^ IF) VF) F >F) I i1FNFN001F)FI>F) '^NONO'FNffoOFIO 'T ff IF) IF) FI O F FN O r-H O FI fF O FN FI FI |F- Ft IF) O FN ff o q FI q q d FI FI FI ON d d> d d> d d> d> O fF r-F n FF o 0« iF)oavvcoooo Fr~oo>F)Oooo O fF IT) O VO FN NO IT) 00 F F-) 00 FI r~ o NO O fn ON 00 On FN FN "Nt q q (F- q OnOnif)NOOOOOO NO>F)IF)IF)ffoN0O'^ON r^FtNOOO'^ON'TlF) ff ff FI FI vd ON FI FI ON O On >o NO On On O FN FI 00 FO Fl FN O -rt oo IT) Ff FN 00 NO fF O Fl FN Tt NO O a ^ to G ^ OFNNOFHTf^FFF FlFFFVOFO'TtF-) FI FF ^ FN ON F-) IF) |F- FN FI 00 00 VO >FN ON fF 00 00 )FN ^ FI 'T pF FN >FN Ff ifnVONOVONDFfFfFfFf VOVOVONOVOVOVOVONO ►— IFFI— lk>l-F)— II— I FFHF‘.^|FSn> « I— II— (if'i-FFS r ^ q > ^ m ^ (S ^ (N v,D VD ^ v>0 VO VO VD VD VD VD I— I >-H nH hH HH ^ NH l-H K.. )-H SZ* ^ CM OS ^ ^ O r4 m ^ ^ IT) VD VD VD VC) VD VD VD VD VO ^ l-H H- 1 "-li. »— • »-H HH >-H HH HH ^ HH HH ^ >> > [F. NO F-l O FN IF) FN n FI FI FI 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 39 Table 4. Weight and Caloric Values of Waste Products from Three Sibling Python Ciirtiis Over a Two-Year Period. Dry Ash-free Calories/ Total Weight Dry Calories/ (Ash-free) Calories Date Sample g % Ash Wt.g g g in Sample Animal No. 650495 15 XII 65 Defec. 0.46 10.95 0.41 5094.06 5720.76 2343.27 9 III 66 Defec. 4.64 31.12 3.20 3483.59 5056.61 16163.86 26 VI 66 Defec. 15.11 29.32 10.68 3653.99 5170.92 55211.79 14 IX 66 Defec. 12.71 29.53 8.96 3517.79 4991.12 44711.11 14 IX 66 Uric A 11.03 5.62 10.41 2645.04 2802.41 29174.79 14 XI 66 Defec. 14.60 35.30 9.45 3084.18 4765.49 45029.03 14 XI 66 Uric A 16.02 2.57 15.61 2734.80 2807.03 43811.50 27 I 67 Defec. 49.12 36.59 31.15 3057.01 4820.63 150162.62 27 I 67 Uric A 54.35 4.85 51.71 2664.96 2800.73 144840.58 23 IV 67 Defec. 42.14 35.07 27.36 3134.35 4828.73 132081.51 23 IV 67 Uric A 43.58 5.54 41.17 2631.54 2785.85 114682.51 18 VI 67 Defec. 19.95 18 VI 67 Uric A 23.55 13 VII 67 Uric A 2.16 25 VII 67 Defec. 34.09 25 VII 67 Uric A 27.30 Animal No. 650496 2 XII 65 Defec. 0.29 10.08 0.26 5324.24 5920.97 1544.03 21 III 66 Defec. 0.76 27.92 0.55 4183.93 5804.62 3179.79 8 VI 66 Defec. 0.67 23.03 0.52 4201.13 5457.92 2814.76 19 VIII 66 Defec. 0.75 30.03 0.52 3660.76 5232.05 2745.57 4 XI 66 Defec. 0.63 25.89 0.47 4020.47 5424.84 2532.90 21 III 67 Uric A 1.37 2.55 1.34 2694.78 2765.23 3691.85 10 IV 67 Defec. 1.85 31.43 1.27 3643.02 5312.53 6739.59 10 IV 67 Uric A 0.41 5.19 0.39 2453.23 2587.39 1005.82 5 VI 67 Uric A 1.55 2.08 1.52 2699.87 2757.19 4184.80 28 VI 67 Uric A 0.70 12 VII 67 Defec. 1.76 12 VII 67 Uric A 0.42 Animal No. 650503 6 XII 65 Defec. 0.41 12.28 0.36 5028.39 5732.27 2061.64 3 V 66 Defec. 8.40 35.34 5.43 3403.81 5264.04 28592.00 3 V 66 Uric A 7.73 3.04 7.50 2709.68 2794.76 20945.83 20 VII 66 Defec. 9.27 28.17 6.66 3724.23 5184.51 34523.61 20 VII 66 Uric A 8.65 4.77 8.24 2668.64 2802.23 23083.74 23 VIII 66 Defec. 6.13 32.88 4.11 3259.58 4855.59 19981.23 23 VIII 66 Uric A 4.35 6.88 4.05 2549.74 2738.03 11091.37 25 I 67 Defec. 10.96 31.57 7.50 3417.19 4993.99 37452.40 25 I 67 Uric A 13.32 2.97 12.92 2735.01 2818.77 36430.33 23 VII 67 Defec. 5.79 23 VII 67 Uric A 8.94 weight by 34.5 pounds which is a gain of one pound for every 1.77 pounds of food or an efficiency of 56 percent. Barton and Allen (1961) gave data for an anaconda, Eunectes murinus, which when received was 16 feet 4 inches and 108 pounds. Over the next 81 months the snake ate 539 pounds of ducks and gained 92 pounds (one pound gain per 5.86 pounds food). This represents an efficiency of 17 per- cent. An African python, P. setae, received at slightly over two feet consumed 148 pounds of food during its seventh, eighth, and ninth years of captivity (37 months) and gained 19 pounds, or 7.8 pounds of food for each pound of body weight gain for an efficiency of 13 percent. Brown (1958) found food efficiencies of 28 percent after one year, 34 percent after two years, and 20 percent after five and six years for Matrix sipedon sipedon. Food efficiencies for Spalerosophis Clifford-: were given by Dmi’el (1967). Data are broken down by age groups and sex. Animals under one year had efficiencies 40 Zoologica: New York Zoological Society [55: 2 Table 5. Energy Budget for Three Sibling Python curtus Over a Two-Year Period Animal No. Dates Days 650495 30 June 66 14 Sept. 66 76 650496 4 Nov. 66 10 Apr. 67 157 650503 6 Dec. 65 3 May 66 148 650503 3 May 66 20 July 66 78 650503 20 July 66 23 Aug. 66 34 650503 23 Aug. 66 25 Jan. 67 155 Budget (in % of total energy available) Growth 27.9 6.1 24.7 28.3 28.6 13.7 Maintenance and Movement 60.3 91.2 66.4 59.2 58.1 78.8 Intestinal Waste 7.1 2.3 5.1 7.4 8.6 3.8 Renal Waste A.l 0.3 3.7 5.0 4.8 3.7 Total Energy 100.0 99.9 99.9 99.9 100.1 100.0 for males and females of 21 percent and 22 per- cent; one- to three-year animals, 26 percent and 33 percent; four- to eight-year animals, 11 per- cent and 23 percent; nine- to 13-year animals, 7 percent and 12 percent. The above data show that, after animals reach a certain age (size?), less energy is expended in growth and more in maintenance. Pope (1962), in discussing the natural su- periority of efficiency in pigs, mentioned that “the domesticated pig attains its size more rapidly than does any other barnyard animal, and on less food, too.” Vanschoubroek et al. (1967) summarized the literature containing data for fully-fed pigs. The values are given as kilogram feed per kilogram gain and range from 3.17 to 4.09. These represent efficiencies of 31.5 percent to 24.4 percent, but the food provided was of optimal quality to promote growth with little non-digestable material. Data cited by Broady ( 1945) for cattle and sheep when calcu- lated as efficiencies give a value of 9.6 percent. The pig with its high food conversion efficiency still is not as efficient as the pythons. There are two probable reasons for the high efficiency of pythons. First, being ectotherms they do not have to expend as much energy in maintaining high metabolic levels (brooding excepted). Sec- ond, because they are rather sluggish except when looking for food they expend less energy in activity. A python in the wild would probably expend more energy in activity since food is not as readily available as under captive conditions. Various aspects of lizard energetics have been considered by some authors. The effects of pro- lactin on growth of adult male Anolis caro- linensis were studied by Licht and Jones (1967). The energetics of food intake and growth were evaluated. Caloric contents of food items were calculated and corrected for fecal losses. These figures were used to calculate average calories assimilated per animal per day. The data re- calculated for control animals show that those weighing about 5.2 grams at 32°C on a 14-hour photoperiod in the spring assimilated 54 to 63 calories per gram-day; 5.7-gram animals at 32°C on a six-hour photoperiod in the winter assimilated 26 to 42 calories per gram-day; and 5.4 gram animals at 32°C on a 14-hour photo- period in the winter assimilated 12 calories to 17 calories per gram-day. Animals in the spring, having normal appetities, show caloric intakes which agree well with the results of Johnson (1966) and McNab (1963). Johnson (1966) dealt with one aspect of the energetics of three species of lizards (Sceloporus undiilatus, S. magister, and Cnemidophorus tigris), namely, assimilation. His data are based on analyses of stomach contents. The weight of food eaten is estimated from these analyses. The caloric value of grasshoppers (5363 calories per gram) as determined by Golley (1961) is taken as representative of all food items. The energy assimilated by a 15-gram S. undidatus, a 22- gram C. tigris, and a 30-gram S. magister is estimated at 0.83 kilocalories, 1.57 kilocalories, and 2.17 kilocalories per day (55 calories, 71 calories, and 72 calories per gram-day), respec- tively. McNab (1963) estimated an energy budget for a 19-gram Sceloporus undidatus and made some comparisons with a Peromyscus manicula- tus of the same weight. The estimate involves these assumptions: the body temperature drops at night; the body temperature is regulated at a mean of 35°C by behavioral means during the day; the lizard is active about one-fifth of the daylight hours; and its active metabolism is about 2.5 times its resting metabolism. Recalcu- lated data of Bartholomew and Dawson (1956) show that a 19-gram lizard uses about 1.12 kilo- calories per day, of which 23 percent (0.26 kilocalories per day) is used in activity. The estimate of active metabolism being 2.5 times 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 41 Standard metabolism may be low (Dawson and Bartholomew, 1958). McNab pointed out that a 19-gram S. occidentalis uses about 0.26 kilo- calories per day for activity compared with a 19-gram Peromyscus maniciilatus which uses about 1.70 kilocalories per day; the mouse thus uses 6.5 times more energy. In spite of the greater energy collected by the mouse, it is un- likely that 6.5 times more energy is needed for food gathering. Surface Area-Weight Relationships Benedict (1932) attempted to relate surface area to weight for a series of snakes. His de- terminations of surface area were obtained by measuring distances from the nose and corre- sponding girths and then taking mean values of these measurements to calculate area. Then, assuming that surface area = K weight®-®^, Benedict proceeded to determine K for each snake. The mean value of K for a series of eight snakes of several species weighing 3.49 kilo- grams to 13.21 kilograms was 12.5 and ranged from 12.0 to 13.2. Values of 14.3 and 14.4 obtained for a 31.80-kilogram python on two separate occasions were discarded by Benedict because they were “probably a little too large.” A value of 17 was obtained for the 5.58-kilogram 1931 python which was the only surface area determined directly from the skin. Benedict com- pared his K values with those of Inaba (1911) which were obtained from five snakes ranging in weight from 48 grams to 109 grams. The values were 19.1, 17.0, 17.5, 18.7, and 19.9, with a mean of 18.6. Benedict attributed the difference between his values and those of Inaba to the fact that Inaba skinned his animals, thereby stretching the skin. However, values of K from the present study range from 23.1 and 24.7 for animals weighing 132.6 grams and 166.1 grams to 10.8 for a 99-kilogram animal. From these data it would seem that K varies inversely with weight and is a constant only within limited ranges of weight. These observa- tions are consistent with those of Meeh ( 1879). He stated that K is a constant only for a group of similarly shaped animals. Benedict (1932) and Inaba (1911) assumed that area is proportional to weight to the 0.67 power. However, the actual relationship for a series of seven pythons was determined by calcu- lating a regression of surface area on weight (text-fig. 21). This resulted in the equation A = 43.16 where 0.549 is significantly dif- ferent from 0.67 at the 95 percent level. Thus, the actual proportionality betvveen surface area and weight must be determined by calculation rather than by assuming that the coefficient of weight is 0.67. Surface Area-Length and Weight-Length Relationships For a given length. Python moliirus is a thicker snake than P. reticulatus. Plots of surface area against length (text-fig. 22) and weight against length (text-fig. 23) show this relation- ship. Data for P. sebae are included in the latter figure which show that its shape is closer to P. moliirus than to P. reticulatus. Physiological and Ecological Implications of the Geographic Distribution of Python moliirus and Python reticulatus Licht and Moberly (1965) found that a tem- perature near 30°C is required for development of the eggs of the green iguana. Iguana iguana. Temperatures a few degrees above and below 30°C resulted in death of the embryos. Their concluding statement that “. . . these results illustrate the need for careful attention to the thermal requirements of the eggs in considera- tion of the ecology and distribution of lizards,” can apply equally to snakes. It becomes of par- ticular significance when applied to the distribu- tion of pythons. For the Indian python. Python moliirus, to have a well developed system of physiological thermoregulation while brooding its eggs suggests that the additional heat sup- plied to the eggs may be needed to prevent them from reach a critically low temperature. The mainland distribution of Python moliirus moliirus includes peninsular India from Sind, West Pakistan, and Punjab in the northwest to Bengal in the northeast. Python m. bivittatus is found over the whole Indo-Chinese subregion. It is recorded as far south as Zinba Chanun, Tavoy district, Burma. It has been collected to the north in the region of Yenping, Fukien, China, in the east and in Yuankiang, Yunnan, China, and Myitkyina, Burma, in the west. Python reticulatus is found on the mainland in southern Burma and Thailand as far north as latitude 18°; to the east as far north as Yen-Bai, North Viet Nam; to the south, throughout Malaysia (Pope, 1935; Smith, 1943). These distributions are shown in text-fig. 24 with some of the specific localities mentioned above plotted on the map. The distribution of these pythons conforms closely with the zoogeographical areas set up by Smith (1931). The area of sympatry corresponds to Smith’s Annam area in the east and Indo-Chinese Great Plain area in the west. North of the area of sympatry is Smith’s Trans- Himalayan Mountainous area. The factor limiting the northern distribution of P. reticulatus may be the critical minimum temperature for the development of its eggs. Unfortunately, data on minimum temperature 42 Zoologica: New York Zoological Society [55: 2 Text-fig. 21. Correlation of surface area with weight of two species of pythons (Python molurus and P. reticulatus). Line and regression equation calculated by method of least squares. requirements of python egg development are scarce. Some indication of the requirements for the hatching of Python setae eggs was given by Joshi (1967). He separated a clutch of 28 eggs into four batches. Five of seven of the eggs kept at 72°F to 84°F (22.2°C to 28.9°C) and 65 percent to 80 percent relative humidity hatched in 52 days. Four eggs kept at 86° F to 90°F (30.0°C to 32.2°C) and 80 percent to 90 percent relative humidity hatched in 49 days. Eggs kept at 70°F to 90°F (21.1°C to 32.2°C) and less than 40 percent relative humidity did not hatch. The last batch kept surrounded by moist soil in a dry sunny place did not hatch, but no temperature or humidity data were given. Although the times that the eggs were at the lower temperatures are not provided, it appears that temperatures as low as 72°F (22.2°C) are not deleterious to the eggs as long as the humidity is fairly high. In addition to better information on tempera- ture requirements of egg development, informa- tion is also needed on climate for the regions where these snakes are found. The distributions of P. molurus and P. reticulatus correspond roughly with the surface temperature regions of Parkins (Espenshade, 1964). Python reticulatus ranges through the area of hot summers and winters, while P. molurus is found also in the regions of hot summer and mild or cool winters (hot = above 20°C; mild = 10° to 20°C; cool = 0°C to 10°C). These data support the hypothesis that distri- bution of these two pythons is limited by egg development temperature. However, additional data are needed to substantiate this hypothesis. General Discussion Colbert, Cowles, and Bogert (1946) de- termined heating rates of alligators by tethering them in the sun and recording their cloacal tem- peratures. They demonstrated a 1°C/1.5 minute (6.6 X 10-1 ° /minute) increase in temperature for a 50-gram alligator and a l°C/7.5 minute (1.3 X 10-1 '/minute) increase for a 13,000- gram alligator with 260 times the body mass. They interpolated to find a l°C/86 hour (1.9 x 10-1 '/minute) temperature rise for a nine- million-gram dinosaur, having 700 times the body mass of the large alligator. In a later paper (Colbert, Cowles, and Bogert, 1947) they de- 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 43 fended objections made to their interpolation. The objectors had pointed out that surface area, mass, and heat capacity must be taken into con- sideration. The recalculated times for a 1°C rise were 67 minutes (1.5 x 10— Vminute) from one person and 66 minutes to 81.5 minutes (1.5-1. 2 X 10-2 °/minute) from the other. The authors admitted their original figure was de- rived incorrectly but insisted that the time would still be as long as several hours rather than the low figures submitted. They claimed that the alligator cannot be treated as a cylindrical, inani- mate mass but that various physiological pro- cesses must be considered. Bartholomew and Tucker (1963) gave an example of what effect physiological processes have on heating and cooling in the agamid lizard, Amphibolurus barbatus. They compared the heating and cool- ing rates of live and dead lizards. The live lizards heated more rapidly than they cooled, thus showing some physiological control. However, the cooling rate of the live lizards was still more rapid than the heating and cooling rates of the dead lizard. This would tend to support the objections raised against the conclusions of Colbert, Cowles, and Bogert. A live animal with physiological control of its heating and cooling rates heats up more rapidly than a dead animal. Therefore, a live animal should also heat up more rapidly than an inanimate model having the same thermal conductivity. The demonstration of physiological thermo- regulation in some lizards (Bartholomew and Tucker, 1963, 1964; Bartholomew, Tucker, and Lee, 1965) and of physiological thermoregula- tion and thermogenesis in pythons (Hutchison, Dowling, and Vinegar, 1966) suggests that mechanisms of physiological thermoregulation occurred in some of the large primitive reptiles and did not originate de novo in mammals and birds. Rodbard ( 1949) discussed the possibility that the large membranous sail-like structures of the Permian reptiles D-imetrodon and Edaphosaurus were used as absorbers of solar radiant energy. An argument for dinosaurs having had some sort of physiological thermoregulation was pre- sented by Russell (1965). He first established that the dinosaurs were intermediate between birds and crocodilians and in many ways closer to birds in their skeletal anatomy. Then assum- ing that the similarities are carried through to their soft anatomy, he suggested that dinosaurs had separate arterial and venous circulations and therefore, some degree of homoiothermy. Cys (1967) refuted Russell’s hypothesis of endothermy in the dinosaurs, stating that its presence is not necessary to explain dinosaur extinction. Instead, Cys maintained that the large size of dinosaurs prevented them from finding hibernation sites. Russell (in a comment at the end of Cys, 1967, p. 267) pointed out that sev- eral dinosaurs were certainly small enough to find hibernation sites. He summarized his com- ments by stating that there appears to be some “flaw” in dinosaur make-up, relating to their extinction, that is independent of size, shape, feeding habits, or systematic position. Proving that the “flaw” is homoiothermy may not be possible, but the hypothesis fits much of the available evidence. The ability of large ectotherms to maintain body temperatures above ambient is not confined to reptiles. Carey and Teal (1966) demonstrated that tuna (big eye, Thunnus obesus, and yellow- fin, T. albacares) can maintain elevated body temperatures with a countercurrent heat ex- change system located in the vascular system of the red muscle masses. The heat exchanger pro- vides a thermal barrier which prevents heat from being carried off by the blood and lost through the gills. Maximum temperatures found were 32°C in a 70 kilogram T. obesus from 20°C water and 26°C in a 12 kilogram T. albacares from 19°C water. The objection that the high internal temperature results from the fish strug- gling on deck was disproved by experiments with curarized fish and measurements of free- swimming fish. Text-fig. 22. Correlation of surface area with length of two species of pythons (Python molurus and P. reticulatus). Lines and regression equations calculated by method of least squares. 44 Zoologica: New York Zoological Society [55; 2 Text-fig. 23. Correlation of weight with length of three species of pythons (Python molurus, P. reticu- latus and P. sebae). Lines determined by method of least squares. P. molurus — = (L3-358)/i4igo P. sebae - W = (L3-210/6155 P. reticulatus — W = (L^-^si)/ 13750 Tied in with the ability to regulate body tem- perature is the ability to sense the temperature of the environment. A temperature sensitive center, with maximum sensitivity at the level of the third ventricle of the brain, was demon- strated in Pseudemys elegans by Rodbard, Samson, and Ferguson (1950). Warming of the area resulted in a rise in blood pressure and cooling resulted in a fall in pressure. Evidence for a temperature control center located in the head was given by Heath (1964); he demon- strated head-body temperature differences in Phrynosoma coronatum with the head 3°C to 5°C higher in partially buried animals and 2°C to 4°C higher in active animals. Emergence of these lizards possibly is dependent on head tem- perature and independent of body temperature. Hammel, Caldwell, and Abrams (1967) demon- strated that the behavioral regulation of body temperature in the blue-tongued skink, Tiliqua scincoides, is dependent on a combination of hypothalamic and other body temperatures. This demonstration was made by implanfing ther- modes across the preoptic region of the brain- stem and heating and cooling this area while the lizard was at environmental temperatures of 15°C or 45°C. Further experiments on Tiliqua scincoides (Cabanac, Hammel, and Hardy, 1967) demonstrated five “warm neurons” which increase their activity with rising brain tempera- ture and three “cold neurons” which increase activity with falling brain temperature. These neurons are located in the preoptic region of the brain. The similarities in temperature sensitivity between the brains of mammals and reptiles point to the possibility of reptilian brains having contained the progenitor of the more finely de- veloped hypothalamic thermostat of mammals. Rodbard (1948) discussed some of the evolu- tionary implications of blood pressure changes in response to body temperature changes. These responses were noted in chickens, rabbits, turtles, and frogs. Rodbard suggested that the first amphibians coming onto land encountered greater diurnal changes in temperature than did the ancestral fish. Thus, the function of hypo- thalamic sensitivity was to adjust metabolic ac- tivity to body temperature changes. This hypo- thesis is supported by the work of Cabanac et al. (1967), Hammel et al. (1967), and Heath (1964). The function of the hypothalamus for the fine control of body temperature probably came with the development of homeothermy in mammals and birds. The thermoregulation of brooding pythons may represent an intermediate step in the development of the latter function for the hypothalamus. Bligh (1966) speculated on the significance of the earlier function of the hypothalamus as a “broad-band” control in mod- ern mammals. He suggests that the remnant of the “broad-band” control, dependent only on thermal sensitivity of the hypothalamus, may act in the case of emergencies as during fever, heat and cold stress, and intense activity. The 24-hour shift in deep body temperature demon- strated in water-deprived camels (Schmidt- Nielsen et al., 1957) may be an example of “broad-band” dominance. Similarly, the diurnal or seasonal heterothermy of various mammals may be derived from the early “broad-band” control (Twente and Twente, 1964). Acknowledgments During the course of the research reported herein, the authors were graduate student and associate professor, Department of Zoology, University of Rhode Island, and curator of reptiles. New York Zoological Park, respectively. They also held respective appointments as visit- ing research fellow and research associate of the New York Zoological Society and adjunct pro- fessor, Department of Zoology, University of Rhode Island. Much of the material included in this report is based upon a dissertation submitted by the senior author in partial fulfillment of the requirements for the Doctor of Philosophy de- 1970] Vinegar, Hutchison, and Dowling: Metabolism, Energetics, and Thermoregulation During Brooding of the Genus Python (Reptilia, Boidae) 45 Text-fig. 24. Distribution of Python molurus and P. reticulatus. Western part of range of P. molurus and island distribution of both species not indicated. 1 — Yenping, China; 2 — Myitkyina, Burma; 3 — Yen- Bai, North Viet-Nam. gree in zoology at the University of Rhode Island. The authors thank the New York Zoological Society and its general director, Mr. William G. Conway, for extending cooperation in supplying facilities and space. Special thanks are given to the following present and former employees of the New York Zoological Park who helped the project in some major way: Robert Brandner, Peter Brazaitis, Sam Dunton, Joel Fisher, Itzchak Gilboa, and William Meng. Acknowl- edgment is given the following individuals and institutions for supplying information oh lengths and weights of pythons: Robert E. Garren, Decatur, Georgia; Frank Groves, Druid Hill Park Zoo, Baltimore, Maryland; James Mizaur, Lincoln Park Zoological Gardens, Chicago, Illinois; Roger Conant, Philadelphia Zoological Garden, Philadelphia, Pennsylvania; Sherman A. Minton, Jr., Indiana University Medical Cen- ter, Indianapolis, Indiana; Gerald S. Lentz, St. Louis Zoological Park, St. Louis, Missouri. Judith Osborne Rebach generously allowed dis- cussion of some of her own unpublished data. This work was supported by NIH research grant GM-10156. Literature Cited Baldwin, F. M. 1930. Oxygen consumption at 20° in certain snakes Pituophis sayi and Lampropeltis getulus holbrooki with some notes on size and seasonal difference. Proc. Iowa Acad. Sci. 35:313-318. Bartholomew, G. A. and V. A. Tucker 1963. 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O. 1969. Studies on the respiration and body tem- perature of Constrictor constrictor. M. S. Thesis. University of Rhode Island, Kings- ton. Rodbard, S. 1948. Body temperature, blood pressure, and hypothalamus. Science, 108:413-415. 1948. On the dorsal sail of Dimetroden. Copeia, 1949(3) :224. Rodbard, S., F. Samson, and D. Ferguson 1950. Thermosensitivity of the turtle brain as manifested by blood pressure changes. Amer. J. Physiol. 160:402-408. Russell, L. S. 1965. Body temperature of dinosaurs and its re- lationships to their extinction. J. Paleontol. 39(3):497-501. Schmidt-Nielsen, K., B. Schmidt-Nielsen, S. A. Jarnum and T. R. Houpt 1957. Body temperature of the camel and its re- lation to water economy. Amer. J. Physiol. 188:103-112. SCLATER, P. L. 1862. Notes on the incubation of Python sebae, as observed in the society’s gardens. Proc. Zool. Soc. London, pp. 365-368. Smith, M. A. 1931. The fauna of British India including Ceylon and Burma. Reptilia and amphibia, vol. 1 — Loricata, Testudines. Taylor and Francis, London, pp. i-xxviii, 1-185. 1943. The fauna of British India including Ceylon and Burma. Reptilia and amphibia, vol. 3 — Serpentes. Taylor and Francis, London, pp. i-xii, 1-583. Stemmler-Morath, C. 1956. Beitrag zur Gefangenschafts- und Fort- pflanzungsbiologie von Python molurus L. Zool. Garten, 21 (5-6): 347-364. Twente, j. W. and j. a. Twente 1964. An hypothesis concerning the evolution of heterothermy in bats. Suomal. Tiedea- kat. Toim., Ser. A, IV, Biol. 91/32, pp. 432-442. (not seen). Valenciennes, A. 1841. Observations faites pendant I’incubation d’une femelle du python a deux raies (Python bivittatus, Kuhl.) pendant les mois de mai et de juin 1841. Comp. Rend., Acad. Sci., Paris, 13:126-133. Vanschoubroek, F., R. De Wilde, and P. Lampo 1967. The quantitative effects of feed restriction in fattening pigs on weight gain, efficiency of feed utilization and back fat thickness. Animal Prod. 9(l):67-74. Vinegar, A. 1968. Brooding of the eastern glass lizard, Ophi- saurits ventralis. Bull. So. California Acad. Sci. 67(l):65-68. Wall, F. 1926. The reticulate python Python reticulatus (Schneider). J. Bombay Nat. Hist. Soc. 31:84-90. Wray, G. O. 1862. (Extract of letter communicated to the secretary of the zoological society). Proc. Zool. Soc. London, p. 108. EXPLANATION OF THE PLATES Plate I Brooding Python molurus bivittatus (NYZS Photo). (Vinegar, Hutchison, Dowling). Plate II Three members of a single brood of Python curtus (William Meng Photo). (Vinegar, Hutchison. Dowling). VINEGAR, HUTCHISON & DOWLING PLATE I METABOLISM, ENERGETICS, AND THERMOREGULATION DURING BROODING OF THE GENUS PYTHON (REPTILIA, BOIDAE). VINEGAR, HUTCHISON & DOWLING PLATE II METABOLISM, ENERGETICS, AND THERMOREGULATION DURING BROODING OF THE GENUS PYTHON (REPTILIA, BOIDAE). NEW YORK ZOOLOGICAL SOCIETY The Zoological Park, Bronx, N. Y. 10460 OFFICERS Laurance S. Rockefeller Robert G. Goelet President Executive Vice-President Chairman of the Executive Committee John Pierrepont Treasurer Henry Clay Frick, II Vice-President Howard Phipps, Jr. Secretary Edward R. Ricciuti Editor & Curator, Publications & Public Relations EDITORIAL COMMITTEE Robert G. Goelet Chairman F. Wayne King Peter R. Marler Ross F. Nigrelli William G. Conway Donald R. Griffin Hugh B. House Joan Van Haasteren Associate Editor James A. Oliver Edward R. Ricciuti George D. Ruggieri, S.J. William G. Conway General Director ZOOLOGICAL PARK William G. Conway . . . Director & Curator, Walter Auffenberg . . . Research Associate in Ornithology Herpetology Hugh B. House .... Curator, Mammalogy Robert A. Brown . . Assistant Curator, Animal James G. Doherty .... Assistant Curator, Departments Mammalogy Joseph A. Davis .... Scientific Assistant to Grace Davall Assistant Curator, the Director Mammals & Birds Emil P. Dolensek Veterinarian Joseph Bell . . Associate Curator, Ornithology John M. Budinger . . . Consultant, Pathology Donald F. Bruning . Assistant Curator, Ornithology Ben Shelly Consultant, Nutrition F. Wayne King .... Curator, Herpetology William Bridges . . . Curator of Publications John L. Behler, Jr Herpetologist Emeritus James A. Oliver . . Christopher W. Coates Nixon Griffis . . . AQUARIUM Director . . Director Emeritus Administrative Assistant John Clark Curator Louis Mowbray . Research Associate in Field Biology Jay Hyman Consultant Veterinarian OSBORN LABORATORIES OF MARINE SCIENCES Ross F. Nigrelli . . . Director and Pathologist Harry A. Charipper . . Research Associate in George D. Ruggieri, S.J. . . Assistant Director Histology & Experimental Embryologist Erwin J. Ernst .... Research Associate in Martin F. Stempien, Jr. ... Assistant to the Estaurine and Coastal Ecolop Director & Bio-Organic Chemist Kenneth Gold Marine Ecologist Eli D. Goldsmith .... Scientific Consultant Jay Hyman Research Associate in William Antopol . . . Research Associate in Comparative Pathology Comparative Pathology Myron Jacobs Neuroanatomist C. M. Breder, Jr. ... Research Associate in Klaus Kallman Fish Geneticist Ichthyology John J. A. McLaughlin . . Research Associate in Jack T. Cecil Virologist Planktonology Martin P. Schreibman . . Research Associate in Fish Endocrinology INSTITUTE FOR RESEARCH IN ANIMAL BEHAVIOR [Jointly operated by the Society and The Rockefeller University, and including the Society’s William Beebe Tropical Research Station, Trinidad, West Indies] Peter R. Marler . Paul Mundinger . Donald R. Griffin . Jocelyn Crane . . Roger S. Payne . Director & Senior Research Zoologist . . . . Assistant Director & Research Associate . . Senior Research Zoologist . . Senior Research Zoologist . . . . Research Zoologist Fernando Nottebohm . . . Research Zoologist George Schaller Research Zoologist Thomas T. Struhsaker . . . Research Zoologist Alan Lill Research Associate O. Marcus Buchanan . . . Resident Director, William Beebe Tropical Research Station ZOOLOGICA SCIENTIFIC CONTRIBUTIONS OF THE NEW YORK ZOOLOGICAL SOCIETY VOLUME 55 • ISSUE 3 • FALL, 1970 PUBLISHED BY THE SOCIETY The ZOOLOGICAL PARK, New York Contents PAGE 3 . A Preliminary Study on the Immobilization of the Asiatic Elephant {Elephas maximus) Utilizing Etorphine (M-99). By C. W. Gray and A. P. W. Nettashinghe. Plates I-II 51 4. Epizootics in Yellowtail Flounder. Limanda jerruginea Storer, in the West- ern North Atlantic Caused by Ichthyophonus, an Ubiquitous Parasitic Fungus. By George D. Ruggieri, S.J., Ross F. Nigrelli, P. M. Powles, AND D. G. Garnett. Plates I-X; Text-figure 1 57 Manuscripts must conform with Style Manual for Biological Journals (American Institute of Biological Sciences). All material must be typewritten, double-spaced. Erasable bond paper or mimeograph bond paper should not be used. Please submit an original and one copy of the manuscript. ZooLOGiCA is published quarterly by the New York Zoological Society at the New York Zoological Park, Bronx Park, Bronx, N. Y. 10460, and manuscripts, subscriptions, orders for back issues and changes of address should be sent to that address. Subscription rates; $6.00 per year; single numbers, $1.50. Second-class postage paid at Bronx, N. Y. Published December 15, 1970 © 1970 New York Zoological Society. All rights reserved. 3 A Preliminary Study on the Immobilization of the Asiatic Elephant (Elephas maximus) Utilizing Etorphine (M-99) C. W. Grayi AND A. P. W. Nettashinghe- ( Plates I-II ) A preliminary study of M-99 for the immobilization of the Ceylonese elephant indicates the effective dosage is approximately twice that used in the African elephant, based on comparative body weights. A dosage rate of 7 to 8 mgs of M-99 was necessary to immo- bilize the Ceylonese elephant as compared to 5 or 6 mgs of M-99 for African elephants of almost double the weight. Introduction IN 1966 A SURVEY of Ccylon’s elephant popu- lation was formally launched by the Office of Ecology and the National Zoological Park of the Smithsonian Institution. Grants for support of this project were awarded to Dr. H. K. Buechner and Dr. I. F. Eisenberg from the Smithsonian Foreign Currency Program and the World Wildlife Fund. As part of the project mission, we were charged with the responsibility of introducing the staff of the Wildlife Depart- ment of Ceylon to the use of M-99 as a pos- sible method of tranquilizing elephants involved in crop damage. Utilization of M-99 would allow troublesome animals to be immobilized, tied, and transported to areas for either release in the wild or recruitment into the domestic elephant population which are still employed in lumber- ing operations in parts of Ceylon. Since virtually no work had been undertaken previously to test the immobilizing effects of M-99 on the Asiatic elephant. Dr. C. W. Gray went to Ceylon rn October 1967 in order to test the effectiveness of the drug, to determine a correct dosage for the Asiatic elephant, and to instruct personnel of the Wildlife Department of Ceylon in the use of pertinent equipment. ’National Zoological Park, Smithsonian Institution, Washington, D.C. 20009. ’Faculty of Veterinary Medicine, University of Cey- lon, Paradeniya, Ceylon. The following report is a summary of six attempted immobilizations on the Ceylon ele- phant. Our operation commenced on October 13, 1967, at Inginiyagala and terminated on October 22, 1967. Study Area The elephant immobilization studies were con- ducted in the Gal Oya region of eastern Ceylon on the shores of a large tank, or reservoir, formed by an earthen dam. The dam was con- structed to provide water power for the gen- eration of electricity and irrigation. The area surrounding the tank (Senanayake Samudra) is heavily wooded and extremely bushy. Every ef- fort was made to locate an elephant in a situa- tion where observation could be continuous from the time of injection until immobilization occurred. Methods All personnel were instructed in the assembly of the projectile dart and the handling of the CapChur rifle. Prior to departure from camp, two projectile syringes were prepared, each con- taining M-99 at a 4 mg dose. The unused darts were emptied at the end of each day in order to eliminate the possibility of product deteriora- tion. Another change in technique was the re- constitution of immobilizing drugs in the field in order to eliminate completely the possibility of product potency loss. This also allowed dosage regulation, depending on the estimated height and weight of the elephant (unpublished data 51 52 Zoologica: New York Zoological Society [55; 3 obtained through a cooperative study of cap- tive elephants, made in 1967 with the staff and students of the University of Ceylon School of Veterinary Medicine, Kandy, permitted us to determine weight based on the estimated height) . Elephant No. 1 The animal was sighted in the afternoon mov- ing southwest from the lake toward the forest. After a ten-minute stalk, the animal was shot in the left gluteal with a projectile dart containing 4 mg of M-99 in a 1-cc syringe from a distance of 20 yards, using a medium-charge cartridge. This dosage was selected due to its effectiveness in the immobilization of the African elephant as reported by Horthoorn and Bligh in their paper “The Use of a New Oripavine Derivative with Potent Morphine-like Activity for the Re- straint of Hoofed Wild Animals” (Research in Veterinary Science, vol. 6, no. 3, July 1965). The dart did not stick in the animal and when it was recovered, the needle was bent at a 45° angle. When the elephant was shot, it wheeled, touched the ground with its trunk, picked up a stick, threw it into the air, and entered the brush, where visual contact was lost. The ele- phant continued moving for 15 minutes, and it was apparent that immobilization was un- successful. Elephant No. 2 This eight-and-one-half-foot male Ceylonese elephant was seen on the lake shore bathing. It left the water and was shot inland with 5 mg of M-99, using a high-powered cartridge from a distance of 30 yards. The syringe was equipped with a heavy-duty needle with a barb. The ani- mal was hit in the gluteal region, turned and entered a small, bushy area 150 yards distant. It made no effort to remove the dart. At 15 minutes after injection, a search was instituted, and the elephant was found leaning with its head against a tree, making paddling movements with the left front foot. There was no response to calling or breaking of small branches. When a larger branch was broken, the elephant turned toward the source of the noise, showing obvious narcosis. At 30 minutes after injection, it had returned to its original position, leaning against the same tree, and again responded to the noise stimulant. At 45 minutes after injection, it did not respond to the breaking of larger branches, but when struck on the trunk with a branch, it wheeled and charged; the tail was lifted but its trunk was limp. When approached at 70 minutes after injection, the elephant moved about ten steps toward the group. It picked up dirt, threw it over its back, indicating a functional trunk; and at this point, observation ceased. On the following day we tracked the elephant from the same bushy area, over a log and down into the forest about a quarter-mile distant. There was no indication of any impairment of gait. It should be noted that dosage rates of M-99 were being increased by 1 mg per elephant, in an effort to reach an immobilizing dosage. Elephant No. 3 A lone male was sighted and stalk was started (PI. I, fig. 1); the elephant was shot 30 minutes after being sighted. A 7 mg dose of M-99 was loaded into a 5cc projectile syringe, and a high- powered load was used from a distance of 35 yards. The dart bounced off the animal and the needle was bent in the shape of an S. Apparently the dart struck the pelvis rather than a muscle mass. The elephant went into the jungle about one-half mile distant, turned, and crossed an open space one-quarter mile further, and went into dense jungle. No further attempt was made to follow the elephant, and the last observation was made 25 minutes after the attempted injection. Elephant No. 4 A male elephant, with an estimated height of six and one-half feet and weight of 6,000 pounds, was sighted. By means of a projectile syringe equipped with a heavy-duty needle, a 6 mg dose of M-99 was injected, using a low- powered load from a distance of 15 yards. The elephant was under constant observation; at seven minutes post injection it sat down in a dog-like attitude, and as it turned, fell re- cumbent. At ten minutes post injection, the ear moved slowly but the trunk was flaccid. This animal was marked with paint on the right hip and measured. Since it was in direct sun, 12 mg of M-285 was injected intramuscularly into the right gluteal region (PI. I, fig. 2). At 20 minutes post injection, 4 mg of M-285 was given intravenously into the ear vein (PI. II, fig. 3). Initial response was movement of the trunk tip, then ear, and front foot; one minute after the intravenous injection, the ear started to fan and foot movement increased; at four minutes, the head was raised. Activity continued, and at four and one-half minutes, the animal made its first attempt to rise; at five minutes, the hind foot was swinging as a prelude to rising (PI. II, fig. 4); and at ten minutes, the animal was on all four feet and moving off with no impairment of gait. It was using the tip of its trunk in searching movements until it entered the jungle (PI. II, fig. 5) . No further observation was possible. Elephant No. 5 A full-grown elephant, with an estimated height of seven and one-half feet and weight of 1970] Gray and Nettashinghe: Preliminary Study on the Immobilization of the Asiatic Elephant 53 6,000 pounds, was injected with a 7 mg dose of M-99, using a low-powered charge from a dis- tance of 20 yards. The animal moved about one and one-half miles, collapsed, and became im- mobile at 16 minutes post injection (the first pedal impediment was noticed at 12 minutes post injection). The head was raised one-half minute following its recumbency but there was no attempt to rise. This elephant was measured and found to be eight and one-half feet tall, making the weight approximately 7,000 pounds (data cited on page 52). It was marked on the right hip and the projectile syringe was removed. At 35 minutes after immobilization, the elephant received 14 mg of M-285 intramuscularly. At 48 minutes after injection, ear movement was noted; at 56 minutes, the elephant got up; and at 60 minutes, it took several steps and sat back down on the hind quarters. At this point it was dark, rain had started, and further observations were not made. Early the following morning, the elephant was seen near the same spot with a normal gait and unimpaired movement and activity. Elephant No. 6 An eight-and-one-half-foot elephant was in- jected with an 8 mg dose of M-99, using a heavy-duty needle in the projectile syringe and a medium load, from a distance of 20 yards. Under continuous observation from time of in- jection, the elephant walked 300 yards and en- tered the jungle, turned to the left for approxi- mately 25 yards, entered a small open clearing, and went down seven and one-half minutes after injection. Measurement of the animal confirmed the height estimate; it was then marked and the dart was removed. At 23 minutes after injec- tion, the elephant received 16 mg of M-285 in- travenously; at 43 minutes, it received 5 mg of M-285. Later it moved by pushing itself in a half-circle, and when it stopped, the elephant had a tree between the front and hind legs. It was able to move its ear, its trunk made search- ing movements, and it made an effort to rise. A 10 mg dose of M-285 was administered intramuscularly, and the elephant was left lying in the shade. There was a breeze blowing, the elephant’s respirations were 9 to 10 per minute and no evidence of cyanosis was seen. Close examination revealed the presence of a perfora- tion in the gluteal region, leading us to believe that this was the same elephant described as No. 2. Darkness prevented further observation, but the animal was seen two miles distant the next morning on the edge of the tank. Observation It seems from this limited study that to im- mobilize the Ceylonese elephant, the require- ment of M-99 is approximately twice that for the African elephant, based on comparative body weight. Pienaar, Niekerk, and Young, in their report on the use of oripavine hydro- chloride in the drug immobilization and mark- ing of wild elephants in the Kruger National Park in describing the effect of M-99 on 31 bull elephants (Journal for Scientific Research in the National Parks of South Africa, No. 9, 1966), indicate an effective dosage level of M-99 combined with acetyl promazine totaling 7 mg or 8 mg for African elephants weighing close to 15,000 pounds, and of 5 mg or 6 mg for elephants weighing from 6,000 to 12,000 pounds. Acknowledgments The authors gratefully acknowledge the ef- forts of Warden Lyn DeAlwis, Wildlife Depart- ment of Ceylon, who furnished the necessary guidance and permission to execute the immo- bilization attempts. We were further assisted by Dr. S. Attapattu, Veterinarian, Zoological Gar- dens at Dehiwala. The immobilization team was composed of Ranger B. Ekanayaka and Mr. Melvin Lockhart. Mr. G. McKay and Mr. Anil Jayasuriya assisted in the photography. Field operations were directed by Dr. John F. Eisen- berg. 54 Zoologica: New York Zoological Society [55: 3 EXPLANATION OF PLATES Plate 1 Plate II Fig. 1. Elephant stalk in progress. (All photo- graphs by Dr. F. Kurt) Fig. 3. Intravenous injection of M-285 using ear vein. the Fig. 2. Intramuscular administration of M-285. Fig. 4. Sitting position immediately prior to gaining feet. re- Fig. 5. Elephant now ambulatory showing no dence of narcosis. evi- GRAY a NETTASHINGHE PLATE 1 FIG 1 .A ■' . A PRELIMINARY STUDY ON THE IMMOBILIZATION OF THE ASIATIC ELEPHANT (ELEPHAS MAXIMUS) UTILIZING ETORPHINE ( M-99 ) GRAY & NETTASHINGHE PLATE 11 FIG. 3 FIG. 4 FIG. 5 A PRELIMINARY STUDY ON THE IMMOBILIZATION OF THE ASIATIC ELEPHANT (ELEPHAS MAXIMUS) UTILIZING ETORPHINE ( M-99 ) 4 Epizootics in Yellowtail Flounder, Limanda ferruginea Storer, in the Western North Atlantic Caused by Ichthyophonus, an Ubiquitous Parasitic Fungus George D. Ruggieri, S.J.,^ Ross F. Nigrelli/ P. M. Powles,- AND D. G. Garnett^ (Plates I-X; Text-figure 1) Yellowtail flounders {Limanda ferruginea) were collected from several areas off Nova Scotia and analyzed for Ichthyophonus. The infection was confined to flounders from the Sable Island Bank and Western Bank. Extensive lesions caused by the fungus were present in the heart, liver, kidney, spleen, gastrointestinal tract, and body musculature. The gills, gall bladder, brain, and testes were mildly infected. All developmental stages of the fungus were observed, although in most histological sections the fungi appear as “resting” or as stages in germination and hyphal development. An analysis of infected fish indicates no relationship to sex and that yellowtails in the size range from 24 cm to 40 cm are more heavily infected. The pathological manifestations of the fungus in the various organs and their significance are discussed. Introduction ICHTHYOPHONUS, the causc of systemic myco- sis in fishes, is characterized macroscopically by the presence of single, multiple, or con- fluent whitish cyst-like lesions in the viscera and in other parts of the body. The disease has been reported in a wide variety of feral and captive fishes of fresh water, brackish water, and marine habitats in cold, temperate, and tropical parts of the world. In every case the causative organ- ism has been identified as a single, pleomorphic species, Ichthyophonus hoferi Plehn and Mul- sow, 1911. Whether or not a single species of fungus is indeed responsible for the disease in all host species remains to be established. This fungus had also been referred to as Ichthyo- sporidium hoferi (Plehn and Mulsow) Pettit, 1911. The generic name Ichthyosporidium Caul- lery and Mesnil, 1905, is now restricted to cer- tain protozoan parasites of fishes and marine invertebrates in the order Haplosporidia, class Sporozoa (Sprague, 1965). Most of the literature on Ichthyophonus deals with sporadic cases. However, recurring inci- ’Osborn Laboratories of Marine Sciences, New York Aquarium, New York Zoological Society, Brooklyn, New York 11224. -Department of Biology, Trent University, Peter- borough, Ontario, Canada. ^Zoology Department, McGill University, Montreal, Canada. dences in epizootic proportions have been re- ported in: the sea herring {Clupea harengus) in the western North Atlantic (see Sindermann, 1961, 1966, and 1970 for reviews) ; the mackerel (Scomber scomber) in the eastern North Atlan- tic (Sproston, 1944); rainbow trout (Salmo gairdneri) in hatcheries in the western United States (Rucker and Gustafson, 1953); and, more recently, yellowtail flounder {Limanda ferruginea) in the western North Atlantic, es- pecially from western Sable Island Bank (Powles, Garnett, Ruggieri and Nigrelli, 1968). The present investigation deals with the pathology of the disease in the yellowtails. It also includes a further analysis of the distribu- tion of the infection in fish collected in 1967 from several areas off Nova Scotia, including the site where the epizootic first occurred in 1966. Material and Methods The yellowtail flounders were collected from the Gulf of St. Lawrence, Banquereau, Middle Ground, Western Bank, and Sable Island Bank (text-fig. 1 .) The sampling routine was as follows: 200 flounders were collected from each area; from each group a sub-sample of 50 specimens preserved in formalin was shipped to the Pathol- ogy Laboratory of the Osborn Laboratories of Marine Sciences, New York Aquarium, for de- tailed microscopic examination of the infection. All fish were examined and the organs of those showing macroscopic evidence of the lesions 57 58 Zoologica: New York Zoological Society [55: 3 were embedded in paraffin, sectioned between 3 and 5 microns, and stained with Harris’ hema- toxylin-eosin. Periodic acid Schiff, Bauer’s chromic acid Schiff, Gram Weigert, Mallory phosphotungstic acid hematoxylin, Masson’s trichrome stain, and Mallory’s modification of the Azan staining method. Observations 1 . Incidence and distribution. Text-figure 1 is a map showing the localities from which the samples were taken. The infection was confined to the Sable Island Bank and the Western Bank, with incidences of 25 percent and 57.4 percent, respectively. The incidence seems to decrease eastward; e.g. yellowtails from south of Sable Island show only a 2.8 percent incidence. Fish from all other areas, including the Gulf of St. Lawrence, appeared to be free of the infection, at least macroscopically. The evidence indicates that the stocks from Sable Island, Banquereau, and St. Lawrence do not mix. It is suggested that the isolation of the disease to the Sable Island area may be related to higher bottom tempera- tures. Whether or not this is a significant factor remains to be determined. An analysis (Table 1) of the fish collected from Western Bank and Sable Island Bank shows the incidence of the disease in relation to size and to the intensity of the infection as seen macroscopically in the liver, heart, kidney, and gastro-intestinal tract. There is no apparent relationship as to sex, although there is some evidence that males ap- pear to be more susceptible. Yellowtails in the size range from 24 cm to 40 cm are more sus- ceptible; the disease appears to be present in proportion to the most abundant sizes, which fits in with the observation that there is little or no size segregation in this species. Further, a pre- liminary analysis (Garnett and Powles) suggests that there is no significant length-weight (co- efficient of condition) relationship between dis- eased and normal fish. A growth and mortality study should be done to make such observations significant. It should be emphasized that the absence of obvious lesions in both younger and older yellowtails as shown in Table 1 does not indicate that they are entirely free of the fungi, since it is possible that deep-seated and isolated “cysts” may be present, e.g. in the brain. 2. Parasite. Because of certain morphological and pathological characteristics, the causative organism was identified as a phycomycete of the genus Ichthyophoniis. Whether or not Ichthyo- phoniis hoferi is the specific agent responsible for the mycosis in the yellowtails can only be determined after a more thorough study of the organism under cultural conditions, by experi- mental infections, and by comparative analysis with isolates from other freshwater and marine hosts. The use as a diagnostic procedure of measurements and descriptive terms for the various stages seen in tissue sections is at pres- ent meaningless. All stages in the development are identifiable, but in most sections the fungi appear as “resting,” often thick-walled cysts (e.g., PI. Ill, fig. 6), or as stages in germination and hyphal development (PI. Ill, fig. 5, and PI. VII, fig. 13) generally characteristic of this type of mycotic infection in fishes. The pres- ence of condia-like bodies in kidney tissue (PL V, fig. 10) is interesting; similar spores have been reported by Sproston (1944) in fungal cultures made from mackerel and their development was also referred to by Reichen- bach-Klinke and Elkan (1965) for Ichthyo- phoniis infection in other marine fishes, but not the herring. 3. Pathology. Extensive lesions caused by the fungus were present in the liver (PL I-III, figs. 2-6), kidney (PI. IV-V, figs. 7-10), spleen (PI. VI, fig. 11), body musculature (PI. VI, fig. 12; PI. VII, fig. 13), heart PI. I, fig. 1; PI. VII, fig. 14; and PI. VIII, fig. 15), gastro-intestinal tract (PL VIII-X, figs. 16-19) and to a lesser degree in the gills, gall bladder, brain, and testes (PI. X, fig. 20). Lesions were not present in the ovary. The absence of the infection in the ovary and the relatively mild pathology in the testes indicates that the disease may have very little effect on potential reproductive ability. As is well known for mycotic diseases, no one tissue change is entirely pathognomic of the fungus infection in fish. In the yellowtails, the lesions are generally characterized by the ab- sence of classical inflammatory responses. How- ever, the lesions involve a great deal of necrosis, especially in those areas showing activities asso- ciated with germination and hyphal growth. In areas where numerous “resting cysts” are pres- ent, the fungi are relatively inert and behave as foreign bodies, i.e. they become surrounded by histiocytes (epithelioid elements), typical of many granulomas (PL V, fig. 9; PI. VI, fig. 11; and PL VIII, fig. 16), or by connective tissue fibers (PL II, figs. 3, 4; PL IV, fig. 7; PL VII, fig. 14; PL VIII, fig. 15; PL X, fig. 20). This is not surprising since similar reactions have been reported for certain mycoses in humans and other mammals. In relatively heavy infections, atrophy effects due to pressure with concomitant necrosis are quite evident in the parenchymal tissue, e.g. liver (PL II, figs. 3, 4) and kidney (PL IV, figs. 7, 8, and PI. V, fig. 9), and in the heart (PL VII, fig. 14, and PL VIII, fig. 15) and body musculature (PL VI, fig. 12). 1970] Ruggieri, Nigrelli, Powles, & Garnett: Epizootics in Yellowtail Flounder 59 Text-figure 1. Map of Western North Atlantic. Yellowtail flounders were collected from the Gulf of St. Lawrence, Banquereau, Middle Ground, Western Bank, and Sable Island Bank. 60 Zoologica: New York Zoological Society [55: 3 Table 1. Macroscopic Analysis of Fish from Western Bank and Sable Island Bank. Total Total Intensity of Infection length number Number Stomach in cm fish Infected Liver Heart Intestine Kidney 16 4 0 17 3 0 18 2 0 19 2 0 20 5 0 21 3 0 22 5 1 + 23 2 0 24 2 1 + + + + + 25 2 1 + + + + + -I- + 26 7 3 + + + + + + + + + + + + + + + + + + -1- + + + + -h + + + + -f 27 7 1 4--1- + + + + + 28 6 2 + + + + + + + + + + -f + + + + + + -h 29 4 3 + + + + + -f + + -h + + + + + + + + + -1- + + + + + 30 8 5 + -h + + + + + + + + + + + + + + -h + + + + + + + + + + + + + + + + + + + + + + + + + -h 31 6 1 + + + 32 3 2 -)- + + + + + -h + -f 33 8 5 + + -f + + + + + + + "h + + + + + + + + + + -f-f + + + + + -f -1- + + + + -h -t- + -f + + 34 3 1 + + + + + 35 5 4 -h + + + + + + + + -f + + -f + + -h + + + + + + + + + + -f 36 4 2 + + + + + + + + -1- + + + + 37 5 3 + + + + + + + + -h + + + + + -f + + + + -f -h + + + + + + + + + + 38 1 0 39 1 1 40 2 2 + + -h + + + + + + + 41 1 0 43 1 1 + + + 44 2 0 46 2 1 + + + + + = less than 5 small cyst-like lesions + + = 6-10 small cyst-like lesions = 10-20 small cyst-like lesions or 1 large confluent patch + + + + = over 20 small cyst-like lesions or several large confluent patches 1970] Ruggieri, Nigrelli, Powles, & Garnett: Epizootics in Yellowtail Flounder 61 Discussion and Summary The yellowtail flounder (Limanda ferriiginea) is the third major species of North Atlantic food flsh to be alfected by recurring epizootics caused by the fungal parasite Ichthyophonus, with inci- dences ranging from 2.8 percent to 57.4 percent. The other two species, referred to above, are the Atlantic herring and mackerel, with inci- dences ranging from 2-80 percent (average 25 percent) and 38-70 percent respectively (Wal- ford, 1958). The disease in the yellowtails ap- pears to be limited to populations in the area of Sable Island off the coast of Nova Scotia but the effects of the infection on growth and on mortality rate are at present unknown. The ab- sence of striking evidence of mass mortalities or fluctuations in the populations of this species in the epizootic regions is surprising. The damage to such vital organs as the heart, liver and kidney is so extensive that there can be no question that homeostasis is affected to the extent that many must succumb directly to the infection, or are made so weak that they become easy prey, or are readily killed off by any drastic change in the physical and chemical characteristics of the environment. The absence of the disease in yellowtails in the Gulf of St. Lawrence is puzzling since this is one of the areas in which epizootics in the herring have been reported almost in a cyclic fashion since 1900 (Sindermann, 1970). Acknowledgments The authors acknowledge the help of the Fish- eries Research Board of Canada and especially the crew of the “A. T. Cameron.” Literature Cited Caullery, M. and F. Mesnil 1905. Sur des haplosporidies parasites de pois- sons marins. C. R. Soc. Biol. 58: 640-643. Pettit, A. 1911. A propos du microorganisme producteur de la Taumelkrankheit: Ichthyosporidhan ou Ichthyophonus. C. R. Soc. Biol. 70: 1045-1047. Plehn, M. and K. Mulsow 1911. Der Erreger der “Taumelkrankheit” der Salmoniden. Zentr. Bakt. Parasitenk. 59: 63-68. Powles, P. M., D. G. Garnett, G. D. Ruggieri, S.J., and R. F. Nigrelli 1968. Ichthyophonus infection in yellowtail flounder (Limanda jerruginea) off Nova Scotia. 1. Fish Res. Bd., Canada 25: 597-598. Reichenbach-Klinke, H. and E. Elkan 1965. The principal diseases of lower verte- brates. Academic Press, New York, i-xii, 600 pp. Rucker, R. R. and P. V. Gustafson 1953. An epizootic among rainbow trout. The Progressive Fish Culturist 15: 179-181. Sindermann, C. J. 1961. Ichthyosporidiiim hoferi. In: Fungi in oceans and estuaries by T. W. Johnson, Jr., and F. K. Sparrow, Jr. Hafner Publ. Co., New York, pp. 557-562. 1966. Diseases of marine fishes. In: Advances in marine biology. Volume 4: 1-89, Aca- demic Press, New York. 1970. Principal diseases of marine fish and shell- fish. Academic Press, New York, i-x, 369 pp. Sprague, V. 1965. Ichthyosporidiiim Caullery and Mesnil, 1905, the name of the genus of fungi or a genus of sporozoans? Syst. Zool. 14: 110-114. Sproston, N. G. 1944. Ichthyosporidiiim hoferi (Plehn and Mul- sow, 1911), an integral fungoid parasite of the mackerel. J. Marine Biol. Assoc. U.K. 26: 72-98. Walford, L. a. 1958. Living resources of the sea. The Ronald Press Co., New York, i-xv, 321 pp. 62 Zoologica: New York Zoological Society [55: 3 EXPLANATION OF PLATES Plate I Plate VI Fig. 1. Gross manifestation of lesions on the heart of a yellowtail caused by the phyco- mycete Ichthyophonus. Fig. 2. Numerous cysts of the parasite in the liver. Plate II Fig. 3. Liver showing numerous “resting cysts.” Note extensive damage and development of connective tissue. Azan. Fig. 4. Area of the liver showing necrosis and dis- tortion of the parenchymal architecture. Azan. Fig. 11. Granulomatous reaction around “resting cysts” in the spleen. Masson’s. Fig. 12. Infection in the body musculature show- ing damage characteristic of Zenker’s de- generation. Mallory’s. Plate VII Fig. 13. Details of stages of germination in the muscle pathway. Hematoxylin-eosin. Fig. 14. Myocardial degeneration caused by the fungal infection. Note extensive connec- tive tissue development resulting in fibroid swelling. Azan. Plate III Fig. 5. Germination and hyphal development in liver with extensive necrosis of the paren- chyma. Note absence of typical inflam- matory reaction. Resting cysts strongly PAS positive. Fig. 6. A typical resting cyst with multi-nucleated plasmodium in the liver. Hematoxylin- eosin. Plate IV Fig. 7. Extensive infection in the kidney with damaged tubular elements. Bauer’s. Fig. 8. Area of infected kidney with extensive necrosis. Gram Weigert. Plate V Fig. 9. Granuloma-like reaction around a “rest- ing cyst” in the kidney. Note the massing of histiocytes and degenerative changes of tubules due to pressure effects. Azan. Fig. 10. Conidial elements of Ichthyophonus seen in the kidney. Cysts surrounded by histio- cytes. Hematoxylin-eosin. Plate VIII Fig. 15. Germination of fungus in myocardium causing necrosis of the heart muscle fibers; granulomatous lesions were also seen in the pericardium. Hematoxylin-eosin. Fig. 16. Submucosa of the stomach showing the pressure effects of the parasite and the massing histiocytes on the mucosa. Masson. Plate IX Fig. 17. Nest of fungal elements in the submu- cosa of the large intestine; the small in- testine was equally infected. Hematoxylin- eosin. Fig. 18. Cysts in the mucosa of the large intestine. The basement membrane has been pene- trated. PAS. Plate X Fig. 19. Multi-nucleated cysts in a capillary of the submucosa of the stomach. Hematoxylin- eosin. Fig. 20. A fungal cyst in the testis. No pathological changes were noted in spermatogonia or spermatids. Hematoxylin-eosin. RUGGIERI, NIGRELLI, POWLES, & GARNETT PLATE 1 FIG. 1 FIG. 2 EPIZOOTICS IN YELLOWTAIL FLOUNDER. LIMANDA FERRUGINEA STORER. I WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS N THE RUGGIERL NlGRELLl, POWLES. & GARNETT PLATE II FIG. 3 FIG. 4 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS RUGGIERI, NIGRELLI, POWLES, & GARNETT PLATE 111 FIG. 6 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC EUNGUS RUGGIERI, NIGRELLI. POWLES, & GARNETT PLATE IV FIG. 8 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER. IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS RUGGIERI, NIGRELLI, POWLES, & GARNETT PLATE V FIG. 10 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHON US, AN UBIQUITOUS PARASITIC FUNGUS RUGGIERI, NIGRELLI, POWLES, & GARNETT PLATE VI FIG. 12 EPIZOOTICS IN YELLOW/TAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS RUGGIERI, NlGRELLl, POWLES, & GARNETT PLATE VII FIG. 14 FIG. 13 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS RUGGIERI, NIGRELLI, POWLES, & GARNETT PLATE VIII FIG. 15 FIG. 16 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS RUGGIERI, NIGRELLI. POWLES, & GARNETT PLATE IX FIG. 17 FIG. 18 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS RUGGIERI, NIGRELLI. POWLES, a GARNETT PLATE X FIG. 20 EPIZOOTICS IN YELLOWTAIL FLOUNDER, LIMANDA FERRUGINEA STORER, IN THE WESTERN NORTH ATLANTIC CAUSED BY ICHTHYOPHONUS, AN UBIQUITOUS PARASITIC FUNGUS NEW YORK ZOOLOGICAL SOCIETY The Zoological Park, Bronx, N. Y. 10460 OFFICERS Laurance S. Rockefeller President John Pierrepont T reasurer Robert G. Goelet Executive Vice-President Chairman of the Executive Committee Henry Clay Frick, II Vice-President Howard Phipps, Jr. Secretary Edward R. Ricciuti Editor & Curator, Publications & Public Relations William G. Conway Donald R. Griffin Hugh B. House EDITORIAL COMMITTEE Robert G. Goelet Chairman F. Wayne King Peter R. Marler Ross F. Nigrelli Joan Van Haasteren Associate Editor James A. Oliver Edward R. Ricciuti George D. Ruggieri, S.J. William G. Conway General Director ZOOLOGICAL PARK William G. Conway . . . Director & Curator, Walter Auffenberg . . . Research Associate in Ornithology Herpetology Hugh B. House .... Curator, Mammalogy Robert A. Brown . . Assistant Curator, Animal James G. Doherty .... Assistant Curator, Departments Mammalogy Joseph A. Davis .... Scientific Assistant to Joseph Bell . . Associate Curator, Ornithology fjjg Director Donald F. Bruning . Assistant Curator, Ornithology c r> i i tz * ■ • F. Wayne King ... Curator, Herpetology Emil P. Dolensek Veterinarian John L. Behler, Jr Herpetologist John M. Budmger . . . Consultant, Pathology William Bridges . Curator of Publications Emeritus Ben Sheffy Consultant, Nutrition AQUARIUM James A. Oliver Director John Clark Curator Christopher W. Coates . . . Director Emeritus honhMovihv&Y . Research Associate inFieldBiology Nixon Griffis .... Administrative Assistant Jay Hyman Consultant Veterinarian OSBORN LABORATORIES OF MARINE SCIENCES Ross F. Nigrelli . . . Director and Pathologist Harry A. Charipper . . Research Associate in George D. Ruggieri, S.J. . . Assistant Director Histology & Experimental Embryologist Erwin J. Ernst .... Research Associate in Martin F. Stempien, Jr. . . . Assistant to the Estaurine and Coastal Ecology Director & Bio-Organic Chemist Kenneth Gold Marine Ecologist Eli D. Goldsmith .... Scientific Consultant Jay Hyman Research Associate in William Antopol . . . Research Associate in Comparative Pathology Comparative Pathology Myron Jacobs Neuroanatomist C. M. Breder, Jr. . . . Research Associate in Klaus Kallman Fish Geneticist Ichthyology John J. A. McLaughlin . . Research Associate in Jack T. Cecil Virologist Planktonology Martin P. Schreibman . . Research Associate in Fish Endocrinology INSTITUTE FOR RESEARCH IN ANIMAL BEHAVIOR [Jointly operated by the Society and The Rockefeller University, and including the Society’s William Beebe Tropical Research Station, Trinidad, West Indies] Peter R. Marler Director & Senior Fernando Nottebohm . . . Research Zoologist Research Zoologist George Schaller Research Zoologist Paul Mundinger • • • • • Assistant Director .j. st^uhsaker . . . Research Zoologist & Research Associate . , ^ „ r ^ ■ Donald R. Griffin . . . Senior Research Zoologist Alan Lill Research Associate Jocelyn Crane .... Senior Research Zoologist O. Marcus Buchanan . . . Resident Director, Roger S. Payne Research Zoologist William Beebe Tropical Research Station a L. ! Z^S' ZOOLOGIC A SCIENTIFIC CONTRIBUTIONS OF THE NEW YORK ZOOLOGICAL SOCIETY VOLUME 55 • ISSUE 4 • WINTER, 1970 PUBLISHED BY THE SOCIETY The zo(k.b©i6^ P^¥k, n ew York THE TuRAL HISTOEY Contents PAGE 5. Gonadotrophin in the Urine of a Pregnant Indian Elephant — A Case Report. By Eho Fujimoto, Natsuki Koto, Tatsuo Imori, and Sanenori Nakama. Plates I-II 73 Index to Volume 55 80 Manuscripts must conform with Style Manual for Biological Journals (American Institute of Biological Sciences). All material must be typewritten, double-spaced. Erasable bond paper or mimeograph bond paper should not be used. Please submit an original and one copy of the manuscript. ZooLOGiCA is published quarterly by the New York Zoological Society at the New York Zoological Park, Bronx Park, Bronx, N. Y. 10460, and manuscripts, subscriptions, orders for back issues and changes of address should be sent to that address. Subscription rates; $6.00 per year; single numbers, $1.50. Second-class postage paid at Bronx, N. Y. Published January 19, 1971 © 1971 New York Zoological Society. All rights reserved. 5 Gonadotrophin in the Urine of a Pregnant Indian Elephant — A Case Report Eho Fujimoto,’ Natsuki Koto,' Tatsuo Imori," and Sanenori Nakama" (Plates l-II ) In 1963, at Takarazuka Zoo, Japan, a young female Indian elephant became pregnant, and in May, 1965, she gave birth to a very large stillborn calf (weighing 133.3 kg, male). The time of conception was problematical, but it was assumed as April or May of 1963, hence the gestation period may have been 24 or 25 months, a little longer than average. Pregnancy diagnosis was attempted during the early and middle gestation period. For exploration, an urinary gonadotrophin was checked by the Friedman and Aschheim-Zondek tests on the whole urine samples collected twice in August 1963. Results showed apparantly positive responses in both tests. However, the samples collected in May and September, 1964, showed negative in the three tests, including a male frog (Rana) reaction which was subjected to the concentrated urine samples. So, probably a gonadotrophic substance may have been excreted in urine of this elephant at some time of the early pregnancy, and this may be more like FSH than LH in its activity. Introduction The zoo at Takarazuka, Hyogo, Japan, has maintained two Indian elephants for sev- eral years. In 1962, the male was 14 years old and the female 15 years old. Mating behavior was first observed in April 1962 and was fre- quently observed during the day and night. The female became pregnant and gave birth in May 1965 to an abnormally large, male stillborn calf in posterior presentation. It weighed 133.3 kg. The time of conception was problematical, but it was assumed that conception occurred in April or May, 1963. Hence, the gestation period was about 24 to 25 months, a little longer than average (Nalbandov, 1964; Parkes, 1956; Perry, 1953). Pregnancy diagnosis was attempted during the early and middle stages of gestation period. Urine samples were examined for gonadotro- phins by the Friedman, Aschheim-Zondek, and male frog tests. The Friedman and Aschheim- Zondek tests on whole urine collected twice in August 1963 were positive, but whole and con- centrated urine samples collected in May and ' Takarazuka Zoolo-Botanical Garden, Takarazuka, Hyogo, Japan. a Department of Surgery and Obstetrics, School of Veterinary Science, College of Agriculture. University of Osaka Prefecture, Sakai, Osaka, Japan. September, 1964, were negative on these three tests. Thus, it appeared that a gonadotrophic sub- stance was excreted in this pregnant elephant’s urine during the third to the fourth month of gestation, but was not present in the urine at the twelfth to thirteenth and the sixteenth to seven- teenth month of a 24 to 25 months gestation period. Materials and Methods Urine samples were collected at three periods of time during the elephant’s pregnancy; in the second and the fourth week of August, 1963; in the first week of May, 1964; and in the second week of September, 1964. As a control, urine from a 17-year-old non-pregnant female ele- phant, which had been raised with another female for years at the Hanshin Park Zoo, Nishinomiya, was collected the fourth week of August, 1963. Urine samples were collected directly with a ladle (PI. I, fig. 2) during urination. Fach urine sample collected had a pH of 8.6 to 8.8. The urine was weakly acidified to pH 5.0 to 6.0 with acetic acid and filtered through both clean ab- sorbent cotton, that had been washed in ethanol and ether and then dried, and paper. The urine was then washed with three volumes of ethyl ether in a large separatory funnel for about three 73 74 Zoologica: New York Zoological Society [55:4 minutes for the purpose of removing possible toxic substances and steroids. The washed urine was placed in a warm water bath of 37 C, for seven to eight minutes. While stirring with a glass rod, a stream of nitrogen gas was blown on the washed urine to drive off the small amount of ether that remained in it. The control urine was treated similarly. A modified Friedman test was done. The urine sample for injection was separated into two equal portions. Each portion was injected into each of two Japanese white rabbits, one young and the other adult. Twenty-four hours before the first injection, the ovaries and uterus of each rabbit were examined by laparatomy. After con- firming that no large, protruding hemorrhagic follicles or corpora hemorrhagica existed on the ovaries and that the uterus was normal, the first half-dose of urine was injected into the ear vein of each rabbit. Twenty-four hours later the second-half dose was given, and autopsy was done 48 hours after the first injection (Table 1). Thirty I.U. of human chorionic gonadotro- phin (HCG) dissolved in saline solution was injected into another rabbit as a positive control. The Aschheim-Zondek test used in this ex- periment was also modified. Each of five imma- ture female rats, Wistar strain, weighing 29 gm to 37 gm was given injections subcutaneously with urine, six times in three successive days, and an autopsy was performed 1 1 6 to 120 hours after the first injection (Table 2). Beside the findings of follicles or corpora hemorrhagica on the ova- ries of each test rat, the weight of the ovaries of test and control rats were compared and re- corded. The urine concentrate was prepared by the addition of 1/20 amounts (v/v) of 20 percent acid-washed kaolin suspension to the urine, as an adsorbing agent, and processed by the method suggested either by Cutler ( 1949) , and Bradbury et al ( 1949). Each 500 ml of whole urine was finally con- centrated to a 25 ml solution according to each method. The concentrated urine samples were used in the frog test, the Eriedman test, and Aschheim-Zondek test. The male Rana nigromaciilata Hallowel (Jap- anese rana frog) was chosen as the test animal for the frog test. Two ml of the above concen- trated solutions was injected into the dorsal lymph sac at each side of five frogs. Thirty I.U. or HCG dissolved in saline solution was injected into a frog for a positive control. The fluid in the cloaca was pipetted every 30 minutes for a two hour period and was examined under micro- Table 1 . Result of the Friedman Test with the Whole Urine of a Pregnant Indian Elephant No. of U vine Samples No. and Body Weight of Rabbit (gm) Ml. of Urine injected (i.v.) 1st 2nd Autopsy Findings in Ovaries (1) in Uterus (2) Judgment 1 1 (1340) 3.5 3.5 ( 1 ) 9 hemorrhagic follicles (2) enlarged & hyperemic positive 2 (2200) 6.0 6.0 ( 1 ) 11 hemorrhagic follicles (2) enlarged & hyperemic positive 9 3 (1330) 3.3 3.3 ( 1 ) 8-9 hemorrhagic follicles (2) enlarged & hyperemic positive 4 (2740) 6.5 6.5 (1) 9-10 hemorrhagic follicles (2) enlarged & hyperemic positive control 5 (2620) 6.5 6.5 ( 1 ) many small follicles (2) small negative (1) Samples: 1 — whole urine, second week, August, 1963 2 — whole urine, fourth week, August, 1963 control — whole urine, non-pregnant, fourth week, 1963 (2) Time of injections: 1st inj.: at 0-hour, 2nd inj.: at 24-hour (3) Time of Autopsy: at 48-hour Fiijimoto, Koto, Imori & Nakama: Gonadotrophin in the Urine of a Pregnant Indian Elephant 75 1970] scope. When examinations at 30, 60, 90, and 120 minutes showed no spermatozoa in the cloacal fluid, a “negative” report was given. Results Whole urine samples 1 and 2, of August 1963, were both positive on the Friedman test as indi- cated by the presence of hemorrhagic follicles on ovaries of the four test rabbits. The whole urine of the control female elephant was nega- tive. In the Aschheim-Zondek test using immature Wistar rats, weighing 29 gm to 37 gm, the positive reaction was weak with whole urine sample 1, but stronger with whole urine sample 2 (Table 2). The average weight increase of ovaries of test rats was 2.4 and 3.5 times, respec- tively, that of the control rat. The concentrated and whole urine samples collected in May and September, 1964, (sample 3 and 4) were nega- tive in the frog test or the other three tests (Table 3). Though urine samples 1 and 2, collected at the third or fourth month of gestation were posi- tive, unfortunately, no further examinations were done in 1963 to follow the excretion pat- tern of gonadotrophic substance in urine of this elephant. Thus it could be concluded that in the third to fourth month of gestation, some gonadotro- phic substance was excreted in this elephant’s urine, and it had disappeared by the twelfth to thirteenth month of gestation. Discussion The details of the behavioral observations throughout estrus, mating, and parturition of this elephant were made in 1966 (Koto and Fuji- moto) . Nalbandov (1964) noted that it has been reported that the elephant (as the mare) forms accessory corpora lutea, but only from about the end of the sixth to the ninth month of the 24 months gestation. He inferred that elephants secrete a gonadotrophic substance similar to the one produced by pregnant mares. So, there seemed to be some difference in time in the secre- tion or excretion of gonadotrophin, between the findings obtained by them, six to nine months, and by us, three to four months. However, the time of conception may probably be hard to judge in most pregnant elephants. Actually in the experiment recorded here, “mating behav- ior” was often observed over a long period of time, apparently even in the pregnant period. True or successful copulation, however, was only observed once at midnight by an attendant (June 16, 1962) and the mating was infertile. Table 2. The Aschheim-Zondek ( Rat) Test on Urine erom a 3-4 Month Pregnant Indian Elephant 1. Technic of Test Day Injection of Urine Morning Noon Evening 1 1st — 2nd 2 3rd 4th 5th 3 6th - - 6 Autopsy (at 1 1 6 to 120 hours after the 1 st injection ) 2. Results of Test No. of rat Volume of Urine per Injection (1st to 6th) No. of Samples 1 2 control Reaction Judgment Reaction Judgment Reaction Judgment 1 0.3 ml each I I 0 2 0.5 " " II 11 0 3 1.0 " " I pos. II pos. 0 neg. 4 1.5 " " II II 0 5 2.0 " " I II 0 ( 1 ) Reaction : 0 — very small follicles only I — large follicles II — hemorrhagic follicles III — hemorrhagic corpora lutea (2) Urine samples; No. 1 — whole urine, second week of August, 1963 No. 2 — whole urine, fourth week of August, 1963 control — whole urine, non-pregnant elephant, fourth week of August, 1963 76 Zoologica: New York Zoological Society [55:4 Table 3. Summary of Results of Tests on the Urine of a Pregnant Indian Elephant Sample No. Time in Gestation, urine collected Urine Test Result Controls 1 3-4 mons. (Aug., ’63) whole Friedman Asch.-Zond. pos. pos. pos. (HCG) neg. (saline) 2 3-4 mons. (Aug., ’63) whole Friedman Asch.-Zond. pos. pos. neg. (urine) neg. (urine) 3 12-13 mons. (May, ’64) concentrated by method (A)* Frog (male) neg. pos. (HCG) neg. (saline) 4 16-17 mons. (Sept., ’64) 1. whole 2. concen’ted by method (A) 3. concen’ted by method (B)** Friedman Asch.-Zond. Frog (male) Friedman Asch.-Zond. neg. neg. neg. neg. neg. pos. (HCG) neg. (saline) * method(A), Cutler method (B), Bradbury et al In this experiment, the time of conception was judged by the following observations. The fe- male began to refuse the male, when he was going to mount her starting in May 1963. She became more gentle and quiet. She never bathed in a water pool in the zoo since the later part of May 1963, although she had previously been very fond of it, even in the cold winter season. The body weight of the fetus, 133.3 kg, was above the average, 70 to 122 kg, of newborn Indian elephants in zoos or circuses. So concep- tion might have occurred a little earlier than April 1963. From the information reported here we can- not determine the nature and time of excretion of a possible gondotrophin in the urine of ele- phants in pregnancy, and we have been unable to find another pregnant elephant for further study. Although it may be presumptuous to spec- ulate from one case on the nature of this gona- dotrophin in the urine of a pregnant elephant, from the results obtained in the Friedman test and Aschheim-Zondek test, it seems more like FSH than LH in its activity. Acknowledgments We express our sincere thanks to Dr. Masa- hiro Nagata, Professor Emeritus, School of Vet- erinary Science College of Agriculture, Univer- sity of Osaka Prefecture, Osaka; and Dr. Senjiro Nishimura of the Misaki Park Zoo, Osaka, Japan, for their kind suggestions. We also grate- fully acknowledge Dr. R. V. Short of the Uni- versity of Cambridge, England; Dr. Erank Schbeck of the Population Council of the Rocke- feller University, New York, New York; and the veterinary staffs at the Portland Zoo, Port- land, Oregon, for their kind advice and sugges- tions. Eurther Dr. Kazunari Akagi of the Hansin Park Zoo, Nishinomiya, Hyogo, Japan, provided a control urine of a non-pregnant elephant to us. Dr. Stephen J. Roberts, Professor of the New York State Veterinary College at Cornell Uni- versity, Ithaca, New York, helped us greatly to complete our manuscript for publication over the language barriers. Professor Nalbandov of University of Illinois, Urbana, Illinois, also en- couraged us for the publication. We all thank these doctors very much for their kind help and encouragement. Literature Cited Bradbury, J. T., Brown, E. S., and Brown, W. E. 1949. Adsorption of urinary gonadotrophins on kaolin. Proc. Soc. Exp. Biol. Med., 71, (1949): 228-232. Cutler, J. N. 1949. An appraisal of the male North American frog (Rana pipiens) pregnancy test with suggested modifications of the original technique. J. Lab. Clin. Med., 34, ( 1949): 554-559. Koto, N., and Fujimoto, E. 1966. On the breeding of an Indian elephant. Jap. J. Zoo & Aquarium (in Japanese), 8, 1-2, (1966): 19-23. Nalbandov, A. V. 1964. Reproductive physiology, second ed., W. H. Freeman & Co., San Francisco and London, 1964. Parkes, a. S. 1956. Marshall’s physiology of reproduction, third ed., ed. by A. S. Parkes, Vol. 1, Part 1, Longmans, Green and Co., London, New York, and Toronto, 1956. Perry, J. S. 1953. The reproduction of the African elephant, Loxodonta africana. Phil. Trans. R. Soc., B. 237, (1953): 93. 1970] Fiijimoto, Koto, Imori & Nakama: Gonadotrophin in the Urine of a Pregnant Indian Elephant 11 Fig. 1: Fig. 2: Fig. 3 Fig. 4 EXPLANATION OF PLATES Plate I “Mating behavior,” incomplete copula- tion — June, 1962. Collection of urine by a ladle. Plate II Parturition and a stillborn calf. “Positive” Friedman reaction on an ovary of a test rabbit, (ca. x 5) FUJIMOTO, KOTO, IMORl & NAKAMA PLATE I FIG. 2 GONADOTROPHIN IN THE URINE OF A PREGNANT ELEPHANT FUJIMOTO, KOTO, IMORi & NAKAMA PLATE II FIG. 3 FIG. 4 GONADOTROPHIN IN THE URINE OF A PREGNANT ELEPHANT 80 Zoologica: Index to Volume 55 [1970] INDEX B brooding, see Python C chromosomes, X and Y, see fish poeciliid E elephant, Asiatic iElepbas maximus), a preliminary study on the im- mobilization of, utilizing etor- phine (M-99), (3) 51-56, Plates I-II elephant no. 1 , 52 elephant no. 2, 52 elephant no. 3, 52 elephant no. 4, 52 elephant no. 5, 52-53 elephant no. 6, 53 methods, 51 observation, 53 study area, 51 pregnant Indian, gonadotrophin in the urine of, a case report, (5) 73-80, Plates I-II, Tables 1-2 discussion, 75 materials and methods, 73 results, 75 Elephas maximus, a preliminary study on the immobilization of, utilizing etorphine (M-99), (3) 51-56, Plates I-II elephant no. 1, 52 elephant no. 2, 52 elephant no. 3, 52 elephant no. 4, 52 elephant no. 5, 52-53 elephant no. 6, 53 methods, 51 observation, 53 study area, 51 energetics, see Python epizootics, see flounder, yellowtail etorphine (M-99), see elephant, Asiatic F fish, poeciliid QXiphophorus macu- latus), sex determination and the restriction of sex-linked pigment patterns to the X and Y chromo- somes in populations of, from the Belize and Sibun Rivers of British Honduras, (1) 1-18, Plates I-II, Text-figure 1, Tables 1-8 discussion, 8-14 material and methods, 2-5 iris pattern, 4-5 macromelanophore pattern, 3-4 red and yellow body and fin patterns, 4 results, 5-8 sex chromosomes of fe- males, 5-8 sex chromosomes of males, 5 summary, 14 flounder, yellowtail QLimanda ierruginea Storer) in the Western North Atlantic caused by Ichthyo- phonus, an ubiquitous parasitic fungus, (4) 57-72, Plates I-X, Text-figure 1, Table 1 discussion and summary, 61 material and methods, 57-58 observations, 58 incidence and distribution, 58 parasite, 58 pathology, 58 fungus, parasitic, see flounder, yellowtail G gonadotrophin, see elephant, pregnant Indian I Ichthyophonus, see flounder, yellowtail L. Limanda Ierruginea Storer, epizo- otics in, in the Western North At- lantic caused by Ichthyophonus, an ubiquitous parasitic fungus, (4) bl-12. Plates I-X, Text-figure 1, Table 1 discussion and summary, 61-72 material and methods, 57-58 observations, 58 incidence and distribution, 58 parasite, 58 pathology, 58 M metabolism, see Python P Python, metabolism, energetics, and thermoregulation during brooding of snakes of the genus (Reptilia, Boidae), (2) 19-50, Plates I-II, Text-figures 1-24, Tables 1-5 general discussion, 42-44 materials and methods, 20-22 results and discussion, 22-42 brooding in various python species, 30-34 Chondropython viridis, 32 Morelia spilotes variegata, 34 Python curtus, 30-32 Python reticulatus, 32-33 Python sebae, 33-34 brooding metabolism of Py- thon moluTus bivittatus, 26-28 discussion of reptile ener- getics, 37-41 egg brooding in various rep- tiles, 34-35 energetics of Python curtus, 35-37 false brooding behavior in a female Python molurus molu- rus, 28-30 heat production-weight cor- relation in snakes, 22-24 heart rates and oxygen pulse, 24-25 metabolic responses to tem- perature change, 26 physiological and ecological implications of the geo- graphic distribution of Python molurus and Python reticu- latus, 41-42 standard metabolism of py- thons, 22 pigment patterns, see fish, poeci- liid S snakes, see Python T thermoregulation, see Python X XiphophoTus maculatus, sex de- termination and the restriction of sex-linked pigment patterns to the X and y chromosomes in popula- tions of a poeciliid fish, from the Belize and Sibun Rivers of British Honduras, (1) 1-18, Plates I-II, Text-figure 1, Tables 1-8 discussion, 8-14 material and methods, 2-5 iris pattern, 4-5 macromelanophone pattern, 3-4 red and yellow body and fin patterns, 4 results, 5-8 sex chromosomes of females, 5-8 sex chromosomes of males, 5 summary, 14 NEW YORK ZOOLOGICAL SOCIETY The Zoological Park, Bronx, N. Y. 10460 OFFICERS Laurance S. Rockefeller Chairman, Board of Trustees Henry Clay Frick, II Vice-President Robert G. Goelet President Howard Phipps, Jr. Chairman, Executive Committee George F. Baker, Jr. Vice-President John Pierrepont Treasurer Augustus G. Paine, II Secretary Edward R. Ricciuti Editor & Curator, Publications & Public Relations William G. Conway Donald R. Griffin Hugh B. House EDITORIAL COMMITTEE Robert G. Goelet Chairman F. Wayne King Peter R. Marler Ross F. Nigrelli Joan Van Haasteren Associate Editor James A. Oliver Edw.ard R. Ricciuti George D. Ruggieri, S.J. William G. Conway General Director ZOOLOGICAL PARK William G. Conway . . . Director & Curator, Walter Auffenberg . . . Research Associate in Ornithology Herpetology Hugh B. House .... Curator, Mammalogy A. Brown . . Assistant Curator, Animal T ^ t. . ^ ^ ‘ Departments James G. Doherty .... Assistant Curator, Joseph A. Davis .... Scientific Assistant to Mammalogy ^ Director Joseph Bell . . Associate Curator, Ornithology Emil P. Dolensek Veterinarian Donald F. Bruning . Assistant Curator, Ornithology John M. Budinger . . . Consultant, Pathology F. Wayne King .... Curator, Herpetology Ben Sheffy Consultant, Nutrition John L. Behler, Jr Herpetologist William Bridges . Curator of Publications Emeritus AQUARIUM James A. Oliver Director John Clark Curator Christopher W. Coates . . . Director Emeritus Louis Mowbray . Research Associate in Field Biology Jay Hyman Consultant Veterinarian OSBORN LABORATORIES OF MARINE SCIENCES Ross F. Nigrelli . . . Director and Pathologist Harry A. Charipper . . Research Associate in George D. Ruggieri, S.J. . . Assistant Director Histology & Experimental Embryologist Paul L Cheung Microbiologist Martin F. Stempien, Jr. . . . As.sistant to the Erwin .1. Ernst .... Research Associate in Director & Bio-Organic Chemist ^ , j Estaurine and Coastal Ecology Eli D. Goldsmith .... Scientific Consultant Kenneth Gold Marine Ecologist William Antopol . . . Research Associate in Research Associate in „ Comparative Pathology „ (^ompaiative Pathology Myron Jacobs Neuroanatomist C. M. Breder, Jr. . . . Research Associate in Klaus Kallman Fish Geneticist Ichthyology John J. A. McLaughlin . . Research Associate in Jack T. Cecil Virologist Planktonology Martin P. Schreibman . . Research Associate in Fish Endocrinology INSTITUTE FOR RESEARCH IN ANIMAL BEHAVIOR [Jointly operated by the Society and The Rockefeller University, and including the Society's William Beebe Tropical Research Station, Trinidad, West Indies] Peter R. Marler Director & Senior Fernando Nottebohm . . . Research Zoologist _ , ,, ,. Research Zoologist George Schaller Research Zoologist Paul Mundmger Assistant Director t,, t c* i, i d ; i • cfe Research Associate Thomas T. Struhsaker . . . Research Zoologist Donald R. Griffin . . . Senior Research Zoologist Alan Lill Research Associate Jocelyn Crane . . . . Senior Research Zoologist O. Marcus Buchanan . . . 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